International Journal of

Molecular Sciences Article

Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD Yujian Chen, Yong Liu *, Jiayu Guo, Tao Tang, Jian Gao, Tao Huang, Bin Wang and Shaojun Liu * Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China; [email protected] (Y.C.); [email protected] (J.G.); [email protected] (T.T.); [email protected] (J.G.); [email protected] (T.H.); [email protected] (B.W.) * Correspondence: [email protected] (Y.L.); [email protected] (S.L.); Tel.: +86-10-6821-3039 (Y.L.); +86-10-6693-1304 (S.L.) Academic Editor: Julian Borejdo Received: 3 May 2016; Accepted: 8 June 2016; Published: 17 June 2016

Abstract: Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120. Keywords: AFAP-120; polyclonal antibody; NINS fragment; AFAP-110; B-cell epitope

1. Introduction Actin filament-associated protein-110kD (AFAP-110), originally found as a substrate and binding partner of Src kinase and protein kinase C alpha (PKCα), is an actin filament-crosslinked protein [1–4]. Subsequently, an alternatively spliced isoform of AFAP-110, actin filament-associated protein-120kD (AFAP-120), is identified. AFAP-110 and -120 (AFAPs) are multi-domain proteins containing two pleckstrin homology domains (PH) for interacting with PKCα [5–7], SH2, and SH3 motifs for binding cSrc [8,9] and carboxy-terminal leucine zipper (Lzip), which mediate AFAP oligomerization [2,10] and interactions with F-actin. AFAP-120 contains an additional neuronal insert (NINS) near the carboxy terminus which does not disrupt the reading frame of the downstream coding sequence [11]. The staining of mouse brain sections with an antiserum against AFAPs demonstrated that AFAPs are widely expressed in embryonic and early postnatal brain regions including the cortex, forebrain, cerebellum, and olfactory bulb [12]. AFAPs expression levels were dramatically decreased in the adult brain, with a high concentration only detected in the olfactory bulb [12]. With Northern and Western Int. J. Mol. Sci. 2016, 17, 942; doi:10.3390/ijms17060942

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forebrain, cerebellum, and olfactory bulb [12]. AFAPs expression levels were dramatically decreased 2 of 9 a high concentration only detected in the olfactory bulb [12]. With Northern and Western blot (using phospho-AFAP-specific antiserum) analysis, it was shown that AFAP-110 is expressed in most tissues, but AFAP-120 is specifically expressed in the nervous system [2,11]. In blot (using phospho-AFAP-specific antiserum) analysis, it was shown that AFAP-110 is expressed neurons, AFAP-120 is enriched in the axon and growth cone, and more abundant and more highly in most tissues, but AFAP-120 is specifically expressed in the nervous system [2,11]. In neurons, tyrosine phosphorylated than AFAP-110 [2,13]. AFAP-120 is enriched in the axon and growth cone, and more abundant and more highly tyrosine So far, it is well-known that AFAP-110 plays a critical role in the regulation of the formation and phosphorylated than AFAP-110 [2,13]. maintenance of actin cytoskeleton [6,14], the function of focal contact [14,15], podosome formation So far, it is well-known that AFAP-110 plays a critical role in the regulation of the formation and and invasion [10,16,17], and cell migration [18]. However, relatively little is known about the maintenance of actin cytoskeleton [6,14], the function of focal contact [14,15], podosome formation and functions of neuronal-specific AFAP-120 in the nervous system, especially about the functions of the invasion [10,16,17], and cell migration [18]. However, relatively little is known about the functions of NINS fragment. In general, a specific antibody is a powerful tool to study the function of protein. neuronal-specific AFAP-120 in the nervous system, especially about the functions of the NINS fragment. Unfortunately, the AFAP-120-specific antibody is still commercially unavailable. Although the In general, a specific antibody is a powerful tool to study the function of protein. Unfortunately, existing anti-AFAP antibody can recognize the AFAP-120 protein, it has several disadvantages when the AFAP-120-specific antibody is still commercially unavailable. Although the existing anti-AFAP used to study the function of the AFAP-120 protein, such as the undistinguished binding to antibody can recognize the AFAP-120 protein, it has several disadvantages when used to study the AFAP-110 and AFAP-120 proteins in immunoprecipitation and immunofluorescence assays. function of the AFAP-120 protein, such as the undistinguished binding to AFAP-110 and AFAP-120 In this study, we predicted B-cell epitopes in the additional NINS fragment of the human proteins in immunoprecipitation and immunofluorescence assays. AFAP-120 protein and selected a peptide containing 16 amino acids as an immunogen. The In this study, we predicted B-cell epitopes in the additional NINS fragment of the human polyclonal antibody against human AFAP-120 was produced by immunizing rabbit with this AFAP-120 protein and selected a peptide containing 16 amino acids as an immunogen. The polyclonal immunogen. The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, antibody against human AFAP-120 was produced by immunizing rabbit with this immunogen. The immunoprecipitation, and immunofluorescence assays. These results suggested that the prepared sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, antibody had an excellent immunoreactivity and would be useful in understanding the function of and immunofluorescence assays. These results suggested that the prepared antibody had an excellent the AFAP-120 protein. immunoreactivity and would be useful in understanding the function of the AFAP-120 protein. Int. J. Mol. Sci. 2016, 17, with 942 in the adult brain,

2. Results 2. Results AFAP-120 Protein ProteinEpitope EpitopePrediction Prediction 2.1. Sequence Analysis and AFAP-120 contain thethe same domains except the AFAP-110 and AFAP-120 AFAP-120 are aremulti-domain multi-domainproteins proteinsthat that contain same domains except NINS (511–594 aa) (Figure 1A). To develop an antibody specifically recognizing AFAP-120, we the NINS (511–594 aa) (Figure 1A). To develop an antibody specifically recognizing AFAP-120, simultaneously predicted the the B-cell epitopes of human NINS using the ABCpred online server and we simultaneously predicted B-cell epitopes of human NINS using the ABCpred online server the BepiPred 1.0 1.0 server. TheThe results showed that and the BepiPred server. results showed thateight eightepitopes epitopes(Figure (Figure1B, 1B, blue blue letters) were predictedby bythe theABCpred ABCpredonline onlineserver, server,and and seven epitopes (Figure letters) by the BepiPred predicted seven epitopes (Figure 1B,1B, redred letters) by the BepiPred 1.0 1.0 server. In those predicted epitopes, a 16aa-peptide sequence (SNHYKYPASAQSVTNT) server. In those predicted epitopes, onlyonly a 16aa-peptide sequence (SNHYKYPASAQSVTNT) waswas the the consensus epitope predicted by tools both (Figure tools (Figure 1B, letters). purple letters). This 16aa-peptide was consensus epitope predicted by both 1B, purple This 16aa-peptide was selected selected as immunogen. as immunogen.

Figure 1.1.Sequence Sequence analysis prediction of AFAP-120 B-cell epitopes. (A) Human Figure analysis andand prediction of AFAP-120 protein protein B-cell epitopes. (A) Human AFAP-110 AFAP-110 (730 aa) and AFAP-120 (814 aa) proteins contain identical SH3 binding domains, two SH2 (730 aa) and AFAP-120 (814 aa) proteins contain identical SH3 binding domains, two SH2 binding binding domains, two homology pleckstrin(PH) homology and a(Lzip) leucine zipper (Lzip) motif. domains, two pleckstrin domains,(PH) and adomains, leucine zipper motif. AFAP-120 contains AFAP-120 contains aninsert additional insert (NINS)by (511–594 aa),splicing; generated by alternative an additional neuronal (NINS)neuronal (511–594 aa), generated alternative (B) B-cell epitopes splicing; (B)sequence B-cell epitopes in the NINS sequence simultaneously predicted by the in the NINS were simultaneously predictedwere by the ABCpred online server and the ABCpred BepiPred online server and the BepiPred 1.0 server. black letters represent the sequence NINS (from 511 1.0 server. The black letters represent theThe sequence of NINS (from 511 to 594 aa).of The blue letters represent epitopes predicted by thethe ABCpred server. letters online represent the epitopes to 594 aa).the The blue letters represent epitopesonline predicted byThe the red ABCpred server. The red predicted by the BepiPred 1.0 server. The by purple represent the common epitope predicted by letters represent the epitopes predicted the letters BepiPred 1.0 server. The purple letters represent both tools. Theepitope black dots represent non-epitopes predicted by both tools. non-epitopes predicted by the common predicted by both tools. The black dots represent both tools.

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2.2. 2.2. Identification Identification and and Purification Purification of of the the Synthetic Synthetic Epitope Epitope Peptide Peptide After After being being synthesized, synthesized, the the 16aa-peptide 16aa-peptide was was identified identified by by mass mass spectrum spectrum (MS). (MS). The The result result showed that the molecular weight of synthetic peptide was 1871.02 Dalton, and its characteristics of showed that the molecular weight of synthetic peptide was 1871.02 Dalton, and its characteristics MS accorded well with the theoretical of MS accorded well with the theoreticalvalue valueofofthis this16aa-peptide 16aa-peptidesequence sequence(Figure (Figure2A). 2A). When When the the synthetic peptide was purified with high performance liquid chromatography (HPLC) (Figure synthetic peptide was purified with high performance liquid chromatography (HPLC) (Figure 2B), 2B), the 1). This the purity purity was was 95.2168% 95.2168% (calculated (calculated by by peak peak area) area) (Figure (Figure 2B, 2B, Table Table 1). This synthetic synthetic peptide peptide was was conjugated with the carrier protein KLH and was then used to immunize a rabbit to produce conjugated with the carrier protein KLH and was then used to immunize a rabbit to produce the the anti-AFAP-120 polyclonal antibody. antibody. anti-AFAP-120 polyclonal

Figure and purification purificationof ofthe thesynthetic syntheticepitope epitopepeptide. peptide.(A) (A) Mass-spectrum graph Figure 2. Identification and Mass-spectrum graph of of the synthetic peptide; (B) Purity identification of the synthetic peptide by HPLC. the synthetic peptide; (B) Purity identification of the synthetic peptide by HPLC. Table Table 1. Purification analysis of synthetic peptide.

No. Retention Time Retention Time 1 8.190 1 8.190 2 8.393 2 8.393 3 8.944 3 8.944 Total Total No.

Peak Area Content (%) Peak Area Content (%) 118,254 2.6289 118,254 2.6289 4,283,028 95.2168 4,283,028 95.2168 96,906 2.1543 2.1543 96,906 4,498,188 100 4,498,188 100

2.3. Antibody to to Human Human NINS NINS Peptide Peptide 2.3. Recognization Recognization of of the the Anti-AFAP-120 Anti-AFAP-120 Antibody To detectwhether whether human NINS could be immunoblotted the anti-AFAP-120 To detect human NINS could be immunoblotted by the by anti-AFAP-120 antibody,antibody, plasmids plasmids pCMV-Flag-human NINS and empty vector pCMV-Flag were transfected into HEK293T pCMV-Flag-human NINS and empty vector pCMV-Flag were transfected into HEK293T cells for 48 h. cells for 48 h. The cells lysed, and the extracted proteins werewith immunoblotted with antibodies The cells were lysed, andwere the extracted proteins were immunoblotted antibodies against AFAP-120 against AFAP-120 and FlagMeanwhile, tag, respectively. Meanwhile, the rabbit was used as and Flag tag, respectively. the rabbit pre-immune serumpre-immune was used asserum a negative control. aThe negative control. The results indicated that over-expressed Flag-tagged human NINS could be results indicated that over-expressed Flag-tagged human NINS could be immunoblotted by immunoblotted by anti-AFAP-120 and anti-Flag antibodies (Figure 3), but not by pre-immune anti-AFAP-120 and anti-Flag antibodies (Figure 3), but not by pre-immune serum (Figure 3). serum (Figure 3). 2.4. Recognization of the Anti-AFAP-120 Antibody to Human Denatured AFAP-120 2.4. Recognization of the Anti-AFAP-120 Antibody to Human Denatured AFAP-120 To detect whether anti-AFAP-120 could recognize the over-expressed human AFAP-120 protein, To detect whether couldAFAP-120 recognize and the pCMV-Flag-human over-expressed human AFAP-120 we transfected plasmids anti-AFAP-120 pCMV-Flag-human AFAP-110 into protein, we transfected plasmids pCMV-Flag-human AFAP-120the and pCMV-Flag-human HEK293T cells, respectively. Forty-eight hours after transfection, cells were lysed, and theAFAP-110 extracted into HEK293T cells, respectively. Forty-eight after transfection, the cells lysed, and the proteins were immunoblotted with antibodieshours against AFAP-120 and Flag tag. were Rabbit pre-immune extracted proteins were immunoblotted with antibodies against AFAP-120 and Flag tag. Rabbit serum was used as a negative control. The results showed that anti-AFAP-120 could react with pre-immune serum was used as a negative control. results showed thatprotein, anti-AFAP-120 the Flag-human AFAP-120 protein, but not with the The Flag-human AFAP-110 althoughcould both react with the Flag-human protein, not with the Flag-human AFAP-110 protein, Flag-tagged human AFAP-120AFAP-120 and AFAP-110 couldbut be recognized by the anti-Flag antibody (Figure 4A). although both Flag-tagged human AFAP-120 and AFAP-110 could be recognized by the anti-Flag

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antibody (Figure Neither over-expressed human nor could be be Neither over-expressed human AFAP-120 nor AFAP-110 couldAFAP-120 be immunoblotted by the pre-immune antibody (Figure 4A). 4A). Neither over-expressed human AFAP-120 nor AFAP-110 AFAP-110 could immunoblotted by the pre-immune serum (Figure 4A). serum (Figure 4A). immunoblotted by the pre-immune serum (Figure 4A).

Figure3. Theanti-AFAP-120 anti-AFAP-120antibody antibody recognized recognized human human NINS NINS peptide. peptide. HEK Figure were Figure 3.3.The The anti-AFAP-120 antibody recognized human NINS peptide. HEK 293T 293T cells cells were were transfectedwith with empty vector pCMV-Flag (Flag) or pCMV-Flag-human pCMV-Flag-human transfected with empty vector pCMV-Flag or NINS (Flag-NINS) (Flag-NINS) transfected empty vector pCMV-Flag (Flag)(Flag) or pCMV-Flag-human NINS (Flag-NINS) plasmids plasmids for The extracted extracted cellwere proteins were immunoblotted immunoblotted against plasmids 4848 h.h. The cell proteins were with antibodies against for 48 h. for The extracted cell proteins immunoblotted with antibodies against AFAP-120 AFAP-120 (anti-AFAP-120) or Flag tag (anti-Flag), respectively. Rabbit serum was AFAP-120 (anti-AFAP-120) or Flag tag (anti-Flag), respectively. Rabbit pre-immune was (anti-AFAP-120) or Flag tag (anti-Flag), respectively. Rabbit pre-immune serum was used as a negative used as a negative control and β-actin as the internal control. Black solid arrows indicate used asand a negative andcontrol. β-actinBlack as the internal Black indicate control β-actin as control the internal solid arrows control. indicate specifically-probed Flag-NINS. specifically-probed Flag-NINS. Black asterisksindicate indicate nonspecific recognization recognization of antibodies. specifically-probed Flag-NINS. Black asterisks nonspecific Black asterisks indicate nonspecific recognization of antibodies.

(A) (A)

(B) (B)

(C) (C)

Figure Recognizationofofanti-AFAP-120 anti-AFAP-120 antibody to denatured human AFAP-120. (A) 293T HEK Figure4.4. Recognization antibody to denatured human AFAP-120. (A) HEK Figure 4. Recognization of anti-AFAP-120 antibody to denatured human AFAP-120. (A) HEK 293T 293T transfected with plasmidspCMV-Flag-human pCMV-Flag-humanAFAP-120 AFAP-120and and pCMV-Flag-human pCMV-Flag-human cells cells werewere transfected with plasmids cells were transfected with plasmids pCMV-Flag-human AFAP-120 and pCMV-Flag-human AFAP-110 48 h. extracted cell proteins were immunoblotted with antibodies against AFAP-120 AFAP-110forfor 48 The h. The extracted cell proteins were immunoblotted with antibodies against AFAP-110 for 48 h. The extracted cell proteins were immunoblotted with antibodies against (anti-AFAP-120) or Flag tagor(anti-Flag) respectively. Rabbit Rabbit pre-immune serumserum was used as a AFAP-120 (anti-AFAP-120) Flag tag (anti-Flag) respectively. pre-immune was used AFAP-120 (anti-AFAP-120) or Flag tag (anti-Flag) respectively. Rabbit pre-immune serum was used negative control and β-actin the internal control. Black solid arrows indicate the corresponding as a negative control andasβ-actin as the internal control. Black solid arrows indicate the as a negative control and β-actin as the internal control. Black solid arrows indicate the AFAP protein; (B) HEK293T lysed andwere 25 µg of whole lysate corresponding AFAP protein;and (B)SH-SY5Y HEK293Tcells and were SH-SY5Y cells lysed and 25cell μg protein of whole cell corresponding AFAP protein; (B) HEK293T and SH-SY5Y cells were lysed and 25 μg of whole cell protein lysate to was subjected to immunoblotting assay with anti-AFAP-120. Pre-immune was subjected immunoblotting assay with anti-AFAP-120. Pre-immune serum wasserum used was as a protein lysate was subjected to immunoblotting assay with anti-AFAP-120. Pre-immune serum was used as control a negative β-actin as the internal control;cells (C) were COS-7 cells were with negative and control β-actin and as the internal control; (C) COS-7 infected withinfected recombinant used as a negative control and β-actin as the internal control; (C) COS-7 cells were infected with recombinantexpressing lentivirusesGFP expressing GFP or (lenti-GFP) or GFP-human (lenti-GFP-AFAP-120) lentiviruses (lenti-GFP) GFP-human AFAP-120AFAP-120 (lenti-GFP-AFAP-120) for 48 h, recombinant lentiviruses expressing (lenti-GFP) or GFP-human AFAP-120 (lenti-GFP-AFAP-120) for immunofluorescence 48 h, and immunofluorescence assay was performed using the anti-AFAP-120 antibody. The and assay wasGFP performed using the anti-AFAP-120 antibody. The cells were for 48 h, and immunofluorescence assay was performed using the anti-AFAP-120 antibody. cells were counterstained with Hoechst33258 Theimages merged(Merge) images (Merge) are overlays of counterstained with Hoechst33258 (Hoechst).(Hoechst). The merged are overlays of the The first cells counterstained with Hoechst33258 the were first three panels. bars equal 30 μm.(Hoechst). The merged images (Merge) are overlays of three panels. Scale barsScale equal 30 µm. the first three panels. Scale bars equal 30 μm.

Next,we wedetected detectedwhether whether the the anti-AFAP-120 anti-AFAP-120 antibody antibody could could recognize recognize endogenous Next, endogenous human human Next, we detected whether the anti-AFAP-120 antibody could recognize endogenous human AFAP-120 proteins in mammalian cell lines. HEK293T and SH-SY5Y cells were lysed and the AFAP-120 proteins in mammalian cell lines. HEK293T and SH-SY5Y cells were lysed and the extracted AFAP-120 proteins in mammalian cell lines. HEK293T and SH-SY5Y cells were lysed and the extracted proteins were subjected to an immunoblotting assay with anti-AFAP-120 and pre-immune proteins were subjected to an immunoblotting assay with anti-AFAP-120 and pre-immune serum, extracted proteins were subjected to an immunoblotting assay with anti-AFAP-120 and pre-immune serum, respectively. The results indicated that, in the equivalent whole cell proteins, AFAP-120 serum, respectively. The results indicated that, in the equivalent whole cell proteins, AFAP-120

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respectively. results indicated that, in the equivalent whole cell proteins, AFAP-120 proteins were proteins wereThe immunoblotted in the SH-SY5Y cells by anti-AFAP-120, but not in the HEK293T cells immunoblotted in the SH-SY5Y cells by anti-AFAP-120, but not in the HEK293T cells (Figure 4B). (Figure 4B). Endogenous human AFAP-120 in the cells could not be immunoblotted by pre-immune Endogenous serum (Figurehuman 4B). AFAP-120 in the cells could not be immunoblotted by pre-immune serum (Figure To 4B). further confirm recognition between the anti-AFAP-120 antibody and human AFAP-120 To further recognition between the anti-AFAP-120 andGFP human AFAP-120 proteins, COS-7 confirm cells were infected with recombinant lentivirusesantibody expressing or GFP-human proteins, were with recombinant lentiviruses expressing GFP-human AFAP-120COS-7 for 48cells h, and theinfected immunofluorescence assay was performed usingGFP the or anti-AFAP-120 AFAP-120 for 48 h, and the immunofluorescence assay was performed using the anti-AFAP-120 antibody. The results indicated that molecules labeled by the anti-AFAP-120 antibody had the same antibody.asThe results indicated that4C). molecules labeled the anti-AFAP-120 antibody same location GFP-AFAP-120 (Figure However, whenbylentiviruses GFP-infected cellshad werethe labeled location as GFP-AFAP-120 (Figure However, when lentiviruses GFP-infected cells were by the anti-AFAP-120 antibody, no4C). immunofluorescence signals were detected (Figure 4C).labeled by the anti-AFAP-120 antibody, no immunofluorescence signals were detected (Figure 4C). 2.5. Reaction of the Anti-AFAP-120 Antibody with Native Human AFAP-120 Protein 2.5. Reaction of the Anti-AFAP-120 Antibody with Native Human AFAP-120 Protein An immunoprecipitation experiment was carried out to test the activity of the anti-AFAP-120 An immunoprecipitation experiment was carried out toprotein. test the Plasmids activity ofpCMV-Flag-human the anti-AFAP-120 polyclonal antibody against the native human AFAP-120 polyclonal antibody against the native human AFAP-120 protein. Plasmids pCMV-Flag-human AFAP-120 and empty vector pCMV-Flag were transfected into HEK293T cells. Immunoprecipitation AFAP-120 andblot empty vectorwere pCMV-Flag wereontransfected into HEK293T cells. Immunoprecipitation and Western analysis performed cell lysates from these transfected cells. As shown and Western blot analysis were performed on cell lysates from these transfected cells. As shown in in Figure 5, Flag-AFAP-120 could be immunoprecipitated by both anti-Flag and anti-AFAP-120. Figure 5, Flag-AFAP-120 be immunoprecipitated by both anti-Flag and anti-AFAP-120. However, it could not becould detected in the pre-immune serum-immunoprecipitated proteins.However, it could not be detected in the pre-immune serum-immunoprecipitated proteins.

Figure 5. Recognization of anti-AFAP-120 antibody to native human AFAP-120 protein. HEK 293T Figure 5. Recognization of anti-AFAP-120 antibody to native human AFAP-120 protein. HEK 293T cells were transfected with plasmids pCMV-Flag-human AFAP-120 (Flag-AFAP-120) or empty vector cells were transfected with plasmids pCMV-Flag-human AFAP-120 (Flag-AFAP-120) or empty pCMV-Flag (Flag). Twenty-five micrograms of whole cell protein lysate was used as input to confirm vector pCMV-Flag (Flag). Twenty-five micrograms of whole cell protein lysate was used as input to the expression of the Flag-AFAP-120 (with anti-Flag) or β-actin (with anti-β-actin) by immunoblotting confirm the expression of the Flag-AFAP-120 (with anti-Flag) or β-actin (with anti-β-actin) by (IB). The rest of cell lysates were incubated with anti-AFAP-120, anti-Flag, or pre-immune serum, immunoblotting (IB). The rest of cell lysates were incubated with anti-AFAP-120, anti-Flag, or respectively. The immunoprecipitated (IP) protein complexes were resolved by SDS-PAGE and probed pre-immune serum, respectively. The immunoprecipitated (IP) protein complexes were resolved by with antibodies against Flag. SDS-PAGE and probed with antibodies against Flag.

3. Discussion Discussion 3. As an an adaptor adaptor protein, protein, one one of of the the functions functions of of AFAP-110 AFAP-110 is in the As is to to localize localize kinases kinases involved involved in the organization of of actin actin cytoskeleton [3,14,16]. In In addition, addition, AFAP-110 AFAP-110 can can affect affect the the integrity integrity of of actin actin organization cytoskeleton [3,14,16]. cytoskeleton through through crosslinking crosslinking actin actin filaments filaments [5–7,10,12]. [5–7,10,12]. The The roles roles of of AFAP-110 AFAP-110 in cytoskeleton in oncogenesis oncogenesis have been examined and found that the loss of AFAP-110 in prostate cancer cells could reduce the rate have been examined and found that the loss of AFAP-110 in prostate cancer cells could reduce the of proliferation and orthotopic tumortumor formation in nudeinmice [19]. For[19]. AFAP-120, previousprevious research rate of proliferation and orthotopic formation nude mice For AFAP-120, indicated indicated that it plays a role in organizing actin structures neuronal differentiation and research that it plays a role dynamic in organizing dynamic during actin structures during neuronal suggested that and AFAP-120 maythat helpAFAP-120 regulate the transition from motile precursor to motile morphologically differentiation suggested may help regulate the transition from precursor differentiated neurons [2]. Those differences imply that AFAP-120 may play different to morphologically differentiated neurons [2]. Those differences imply that AFAP-120 roles may from play AFAP-110 in the nervous system. In order to further uncover the functions of the AFAP-120 protein, different roles from AFAP-110 in the nervous system. In order to further uncover the functions of thea highly sensitive andaspecific AFAP-120 is indispensable. However, to now, the AFAP-120 protein, highly antibody sensitive against and specific antibody against AFAP-120 is up indispensable. anti-AFAP-120 antibody is still commercially unavailable, which may hinder the function research However, up to now, the anti-AFAP-120 antibody is still commercially unavailable, which may for AFAP-120. hinder the function research for AFAP-120.

The use of KLH and bovine serum albumin conjugated peptides to prepare anti-peptide antibodies has increased dramatically in recent years. Compared with using proteins as antigens, using synthetic peptides as antigens has the advantages of ready availability and the ease of

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The use of KLH and bovine serum albumin conjugated peptides to prepare anti-peptide antibodies has increased dramatically in recent years. Compared with using proteins as antigens, using synthetic peptides as antigens has the advantages of ready availability and the ease of producing the anti-peptide antibody specifically against protein isoforms or site-specific phosphorylated proteins [20]. Therefore, it is feasible that the use of synthetic peptides derived from the NINS fragment to generate an antibody against AFAP-120 protein. Using experimental methods to characterize epitopes is time-consuming and demands a large amount of resources. The availability of epitope prediction methods can aid experimenters in simplifying this procedure. The ABCpred online software and the BepiPred 1.0 server are two popular B-cell epitopes prediction tools. To obtain high sensitivity, specificity, and positive prediction values, we simultaneously used the ABCpred online serve and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of the human AFAP-120 protein [21,22], and found a 16aa-peptide sequence (SNHYKYPASAQSVTNT) was the consensus epitope predicted by both tools. This 16aa-peptide was chemically synthesized and used as an immunogen to immunize rabbits. After an antiserum was produced successfully, the specificity and sensitivity of the developed AFAP-120 antibody were detected. The results showed that this antibody could react with the over-expressed and endogenous AFAP-120 protein under a denatured condition, but not with the AFAP-110 protein. Moreover, native AFAP-120 protein was also verified to be recognized by this AFAP-120 antibody with immunoprecipitation. 4. Materials and Methods 4.1. Plasmid Constructs The DNA sequence encoding the human AFAP-110 protein was amplified by PCR from the human Schwann cell cDNA library (Beijing, China). The sense primer was 5’-CCGC TCGAGCGGATGGAAGAGTTAATAGTTGAACT-3’, including a XhoI site, and the antisense primer was 5’-CCCAAGCTTGGGGTCCCGTTCTTCAATT-3’, including a Hind III site. The cDNA sequence encoding the human AFAP-120 protein was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and was amplified by PCR using the same primer as described above. Flag-AFAP-110 and Flag-AFAP-120 were constructed by inserting a PCR amplified fragment into the pCMV-Flag vector. The DNA sequence encoding the 84 amino acids of human NINS was amplified by PCR from the plasmid pCMV-Flag-AFAP-120 and was then inserted into the pCMV-Flag vector. The inserted fragment sequences in recombinant plasmids were verified by DNA sequencing (Sangon Biotech Co., Ltd., Shanghai, China). 4.2. Sequence Analysis and B-Cell Epitopes Prediction of the AFAP-120 Protein Firstly, the amino acid sequences of the human AFAP-120 and AFAP-110 proteins were aligned with DNAMAN software (Lynnon Biosoft, San Ramon, CA, USA), and the unique sequences in the AFAP-120 protein were found. The ABCpred online server (http://www.imtech.res.in/raghava/ abcpred/) [21] and the BepiPred 1.0 server (http://www.cbs.dtu.dk/services/BepiPred/) [22] were used to predict B-cell epitopes in this unique sequence of the AFAP120 protein, respectively. The ultimate consensus epitope predicted by both tools was synthesized (Sangon, Shanghai, China) and used as an immunogen. 4.3. Immunization and Production of the AFAP120-Reactive Rabbit Polyclonal Antibody One male rabbit (2.5kg) was injected subcutaneously with the immunogen in Freund’s complete adjuvant (FCA) (Sigma, St. Louis, MO, USA) and Freund’s incomplete adjuvant (FIA) (Sigma, St. Louis, MO, USA) in 2-week intervals. The primary immunization consisted of 800 µL immunogen (1 µg/µL, dissolved in PBS) mixed with an equal volume of FCA. For the subsequent immunizations, 400 µL (1 µg/µL, dissolved in PBS) of the immunogen was mixed with an equal volume of FIA. After 4

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immunizations, the antiserum was harvested and subjected to affinity purification (ABclonal Biotech, Shanghai, China). Rabbit serum collected before the day of the first immunization was applied as a negative control. 4.4. Cell Culture and Transfection HEK293T, SH-SY5Y, and COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen, Waltham, MA, USA), 2 mM glutamine, and 1% penicillin/streptomycin (Sigma, St. Louis, MO, USA) in a 5% CO2 atmosphere at 37 ˝ C. Transfections were performed with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. 4.5. Immunoprecipitation Cells were harvested at 48 h post-transfection and lysed respectively in IP Lysis Buffer (Thermo, Waltham, MA, USA) (25 mM Tris¨ HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). After the protein concentration of each sample in triplicate was determined using the BCA Protein Assay Kit (Thermo, Waltham, MA, USA), the sample (1 mg) were incubated with 3 µg rabbit anti-Flag polyclonal antibody (MBL, Woburn, MA, USA) or 3 µg rabbit anti-AFAP-120 polyclonal antibody in 1 mL IP Lysis Buffer for 8 h at 4 ˝ C, and the immune complexes were precipitated with 20 µL Protein A/G Plus-agarose (Roche, Basel, Switzerland). The immunoprecipitates were then separated by 12% SDS–polyacrylamide gel electrophoresis. 4.6. Immunoblotting Cells were harvested at 48 h post-transfection and lysed in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS) containing a protease inhibitor cocktail. The immunoprecipitates or cells extract proteins were separated by 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane (Osmonics, Minnetonka, MN, USA) using a semidry blotting apparatus (Bio-Rad, Hercules, CA, USA). The membranes were first blocked with 5% skimmed milk in washing buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.6) at room temperature for 1 h and incubated first with primary antibodies at 4 ˝ C for overnight and then with appropriate horseradish peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h after a few washes. The blots were visualized using an enhanced chemiluminescence immunoblotting detection kit (GE Healthcare, Buckinghamshire, UK). 4.7. Immunofluorescence COS-7 cells were infected with recombinant lentiviruses expressing GFP or GFP-human AFAP-120 (Genechem, Shanghai, China). Forty-eight hours after infection, cells were fixed with 4% paraformaldehyde at room temperature for 30 min and permeabilized in 0.3% Triton X-100. The samples were incubated with the rabbit anti-AFAP-120 polyclonal antibody at 1:2000 dilution at 4 ˝ C for overnight, then stained with a TRITC-conjugated secondary antibody (Abcam, Cambridge, MA, USA) at room temperature for 1 h. The cells were further counterstained with 1 µg/mL Hoechst33258 (Sigma, St. Louis, MO, USA) in PBS and visualized using a confocal laser scanning microscope (Leica, Wetzlar, Germany). 5. Conclusions In summary, to develop an antibody recognizing human AFAP-120, we aligned the amino acid sequences of the human AFAP-120 and AFAP-110 proteins, and a unique NINS fragment in the AFAP-120 protein was found. B-cell epitopes in the NINS sequence was simultaneously predicted by the ABCpred online server and the BepiPred 1.0 server. A 16aa-peptide sequence screened out by both

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tools was chemically synthesized and used as an immunogen to develop a polyclonal antibody against AFAP-120. The combined results of immunoblotting, immunoprecipitation, and immunofluorescence analyses demonstrated that the AFAP-120 antibody could react with denatured and native human AFAP-120 proteins, but not with the AFAP-110 protein, which may be a useful tool for studying the functions of the AFAP-120 protein. Acknowledgments: This work was supported by grants from the Chinese National Natural Science Foundation (No. 81471155 and No. 81370051). Author Contributions: Yong Liu and Shaojun Liu conceived and designed the experiments; Yujian Chen, Jiayu Guo, Tao Tang, Jian Gao, Tao Huang, and Bin Wang performed the experiments; Yong Liu and Yujian Chen analyzed the data; Yong Liu and Yujian Chen wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations AFAP-120 AFAP-110 NINS PKCα Lzip MS HPLC FCA FIA SDS-PAGE IB IP

actin filament-associated protein-120kD actin filament-associated protein-110kD neuronal insert protein kinase C alpha leucine zipper mass spectrum high performance liquid chromatography Freund’s complete adjuvant Freund’s incomplete adjuvant SDS–polyacrylamide gel electrophoresis immunoblotting immunoprecipitation

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Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.

Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and conta...
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