2099
Theranostics 2016, Vol. 6, Issue 12
Ivyspring
Theranostics
International Publisher
Research Paper
2016; 6(12): 2099-2113. doi: 10.7150/thno.16587
Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy Jinxie Zhang1,2,*, Xudong Zhang 1,2,3,*, Gan Liu1,2,*, Danfeng Chang1,2, Xin Liang2, 4, Xianbing Zhu1,2, Wei Tao1,2 and Lin Mei1, 2 1. 2. 3. 4.
School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China; Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA; Department of Pharmacological and Physiological Science and Center for Neuroscience, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
* These authors contributed equally to this work. Corresponding authors: Lin Mei: Tel/Fax: +86 75526036736, E-mail: [email protected]. Xudong Zhang: Tel/Fax: +1(314) 882-9360, E-mail: [email protected] © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions.
Received: 2016.06.24; Accepted: 2016.07.30; Published: 2016.09.12
Abstract The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine. Key words: Nanomedicine; Endocytosis; Autophagy; Exocytosis; Protein nanocapsules.
Introduction Most human diseases, including cancer, diabetes and neurodegenerative diseases, are caused by mutations in a class of specific genes. Mutations in these genes can induce the transcription of dysfunctional or nonfunctional proteins that cause pathogenesis. Protein therapy is one of the safest and most direct approaches for the treatment of disease by replenishing functional proteins within unhealthy cells [1]. However, the poor stability and low cellular
permeability of the proteins makes protein therapy difficult to apply clinically [2]. In recent years, a novel delivery platform based on nanocapsules was developed to deliver proteins to target cells. Nanocapsules are designed to consist of a protein core and a thin permeable degradable polymeric shell [3-5]. Once the degradable nanocapsules enter the cells, their shells break down and release the core proteins into the cytoplasm [5]. http://www.thno.org
2100
Theranostics 2016, Vol. 6, Issue 12 Including the newly designed protein nanocapsules, many biodegradable nanocarried particles have been developed, such as PLGA(poly(lactic-co-glycolic acid)-based nanoparticles, micelles, chitosan and microgels [6-10]. Most of these nanocarriers are used as drug nano-vehicles and have promising effects for drug delivery, such as a long lifecycle in the blood stream, targeting to specific cells with ligands on the surface of the particles or cells and multiple drug targets [7, 11]. These designed nanocarriers must enter the cells and release the drugs to the target cells to execute their missions. Most nanoparticles enter the target cells through endocytosis [12]. The main endocytosis pathways are either clathrin dependent or clathrin independent [12]. The clathrin independent pathways include the micropinocytosis, caveolin dependent and caveolin independent (Arf-6, Flotillin, Cdc42 and RhoA- dependent) pathways [12]. After crossing the cell membrane, the nanoparticles are sequestered in vesicles. These vesicles are transported to early endosomes (EEs) and late endosomes (LEs) and finally fuse with lysosomes [12, 13]. Most developed nanoparticles, including PLGA-based nanoparticles, micelles, chitosan and microgels, have been shown to traffic in this classic endocytosis pathway. Modification of nanoparticles with various ligands, such as histidine, detergent and cell membrane penetrating peptides, enhances the nanoparticles’ escape from the endo-lysosome system [14-16]. The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy, which is responsible for the transport of various macromolecules between different organelles in the cells [17-19]. The crosstalk between endocytosis, exocytosis and autophagy makes these networks much more complex. Other endocytotic pathways exist in addition to the EEs-LEs-lysosome pathway. Recycling of the endosome system is responsible for delivering protein receptors back to the cell membrane [12]. The exocytosis pathway is responsible for the transportation of mature proteins from the Golgi body to the cell membrane or for secreting the proteins out of the cells [19]. Autophagy is a cellular process that engulfs cellular components, such as cytoplasm, damaged mitochondrial, aggregated proteins and invading pathogens [18]. Moreover, there are multiple pathways that exist in endocytosis, recycling endocytosis, exocytosis and autophagy. However, most studies focus on the intracellular trafficking and, accordingly, the design of nanocarriers is limited to the EEs-LEs-lysosome pathway. The trafficking pathways such as recycling endosomes, exocytosis and autophagy of the nanoparticles in the cells are poorly investigated and
unknown. The Rab protein family consists of GTPases/GTP- binding proteins that are evolutionary conserved from yeast to humans. Rab proteins have various cellular functions, including protein trafficking, transmembrane signal transduction and the fusion of vesicles between different organelles [20]. More than 70 human Rab proteins have been identified, and the functions of 30 Rab proteins have been identified [21]. Most of these Rab proteins are involved in the trafficking of vesicles in and across endocytosis, recycling endocytosis and exocytosis pathways [22]. Rab5 and Rab7 have been well studied and have become the most important organelle identity markers of EEs and LEs in the endocytic pathway, respectively [22]. To investigate in detail, the trafficking pathways of protein nanocapsules, we used these Rab proteins as vesicle markers to track the intracellular travel pathway of nanoparticles. The crosstalk between the endocytosis and exocytosis pathway with autophagy has also been extensively investigated, through the manipulation of the exocytosis and autophagy pathways with inhibitors such as 3-MA (autophagy inhibitor) and CQ (lysosome inhibitor), which block the degradation of the protein nanocapsules. The effects of the protein nanocapsules may extensively improve when combined with drugs that can block the autophagy and exocytosis pathways.
Materials and methods Materials Bovine serum albumin, dimethyl sulphoxide (DMSO), Chloroquine (CQ), and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-acryloxysuccinimide (NAS), acrylamide (AAm), N, N-methylene bisacrylamide (BIS), ammonium persulphate (APS), N, N, N’, N’-tetramethylethylenediamine (TEMED) and Fluorescein isothiocyanate (FITC) were purchased from Aladdin Industrial Co. LTD. (Shanghai, China). N-(3-Aminopropyl) methacrylamide hydrochloride was purchased from Polymer Science, Inc. All other chemicals of the highest quality were commercially available and used as received. Lyso-Tracker Red was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, Clathrin, Caveolin were from Cell Signaling Technology. The antibody against LAMP1 was from Santa Cruz Biotechnologies, and the antibody against β-actin was obtained from Abmart.
Plasmid and transfection The EGFP-LC3 and DsRed-LC3 plasmids were created in our laboratory. The DsRed-Rab5, http://www.thno.org
2101
Theranostics 2016, Vol. 6, Issue 12 DsRed-Rab7 and Flag-Rubicon plasmids were purchased from Addgene. The KSHV Flag-vBcl-2 plasmid was kindly provided by Professor Beth Levine from the University of Texas Department of Medicine. The Rab1-37 genes in the T Vector were from Professor Jiahuai Han’s Lab and sub-cloned into EGFP-C1 and DsRed-C1, respectively. All plasmids were confirmed by automated DNA sequencing. Cells were transiently transfected with the plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
FITC-label nBSA at 37 ℃ for 20 h. For lysosome detection, the cells were incubated with Lyso-Tracker Red for 1 h. Then, the cells were washed with PBS three times and fixed with 4% paraformaldehyde for 20 min. Cells were stained with DAPI for 15 min and washed three times with PBS. Confocal microscopy was performed with a FLUO-VIEW laser scanning confocal microscope (Olympus, FV1000, Olympus Optical, Japan) in sequential scanning mode using a 60~100×objective. The operation processes were similar to reference [23].
Synthesis of nBSA
Autophagy assays
The synthesis procedures were similar to reference [4]. First, 20 mg of BSA in 4 ml of pH 8, 50 mM sodium carbonate buffer was reacted with 0.5 mg N-acryloxysuccinimide in 50 ml DMSO for 2 h at room temperature. Then, the reaction solution was thoroughly dialysed against pH 7.0, 20 mM phosphate buffer. Using 10 mg acryloylated BSA solution at 1 mg/ml, radical polymerization from the surface of the acryloylated protein was initiated by adding APS dissolved in deoxygenated and deionized water and TEMED into the test tube. A specific amount of N-(3-Aminopropyl) methacrylamide (Apm), AAm and BIS (molar ratio BSA/ Apm/ AAm/ BIS/ Aps/ TEMED = 1 / 500 / 2500 / 300 / 300 / 1200) dissolved in deoxygenated and deionized water was added to the test tube over 60 min. The reaction was allowed to proceed for another 60 min in a nitrogen atmosphere. Finally, dialysis was used to remove monomers and initiators. The unmodified BSA was removed using ion exchange chromatography.
Cells were transfected with EGFP-LC3 under the indicated conditions and then fixed in 4% paraformaldehyde. The percentages of cells with fluorescent dots representing EGFP-LC3 translocation were counted by confocal microscopy as described previously [24-26]. LC3II protein level was detected using an anti-LC3 antibody.
Characterization of nBSA Ion exchange chromatography was performed with a DEAE Sephadex A-25. Gel electrophoresis results were quantified with ImageQuant LAS4000 after agarose gel electrophoresis in 0.7% agarose gels. TEM images of nanocapsules were obtained with a Philips EM120 TEM at 100000X. Before observation, nanocapsules were negatively stained using 1% pH 7.0 phosphotungstic acid (PTA) solution. Zeta potential and particle size distribution were measured with a Malvern particle sizer Nano-ZS.
Cell culture MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS).
Cellular uptake of nBSA FITC was used as a model fluorescent molecule and was formulated in nBSA. Non-transfected or DsRed-Rab1-38 were incubated with 1 mg/mL
Immunoblotting Immunoblotting analysis was performed as previously described [24, 27]. In brief, cell lysates were resolved on 12% SDS-PAGE and analyzed by immunoblotting using an LC3 antibody, followed by enhanced chemiluminescence (ECL) detection (Thermo Scientific).
Immunofluorescence assay Briefly, the cells were incubated with the following primary antibodies: EEA1, Clathrin, LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, and Caveolin. TRITC and FITC labeled secondary antibodies were used to detect the primary antibodies.
Statistical methodology All results are reported as the mean ± S.D of three independent experiments. Comparisons were performed using two-tailed paired Student’s t tests (* P
Theranostics 2016, Vol. 6, Issue 12
Ivyspring
Theranostics
International Publisher
Research Paper
2016; 6(12): 2099-2113. doi: 10.7150/thno.16587
Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy Jinxie Zhang1,2,*, Xudong Zhang 1,2,3,*, Gan Liu1,2,*, Danfeng Chang1,2, Xin Liang2, 4, Xianbing Zhu1,2, Wei Tao1,2 and Lin Mei1, 2 1. 2. 3. 4.
School of Life Sciences, Tsinghua University, Beijing 100084, P.R. China; Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, NC 27695, USA; Department of Pharmacological and Physiological Science and Center for Neuroscience, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
* These authors contributed equally to this work. Corresponding authors: Lin Mei: Tel/Fax: +86 75526036736, E-mail: [email protected]. Xudong Zhang: Tel/Fax: +1(314) 882-9360, E-mail: [email protected] © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions.
Received: 2016.06.24; Accepted: 2016.07.30; Published: 2016.09.12
Abstract The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine. Key words: Nanomedicine; Endocytosis; Autophagy; Exocytosis; Protein nanocapsules.
Introduction Most human diseases, including cancer, diabetes and neurodegenerative diseases, are caused by mutations in a class of specific genes. Mutations in these genes can induce the transcription of dysfunctional or nonfunctional proteins that cause pathogenesis. Protein therapy is one of the safest and most direct approaches for the treatment of disease by replenishing functional proteins within unhealthy cells [1]. However, the poor stability and low cellular
permeability of the proteins makes protein therapy difficult to apply clinically [2]. In recent years, a novel delivery platform based on nanocapsules was developed to deliver proteins to target cells. Nanocapsules are designed to consist of a protein core and a thin permeable degradable polymeric shell [3-5]. Once the degradable nanocapsules enter the cells, their shells break down and release the core proteins into the cytoplasm [5]. http://www.thno.org
2100
Theranostics 2016, Vol. 6, Issue 12 Including the newly designed protein nanocapsules, many biodegradable nanocarried particles have been developed, such as PLGA(poly(lactic-co-glycolic acid)-based nanoparticles, micelles, chitosan and microgels [6-10]. Most of these nanocarriers are used as drug nano-vehicles and have promising effects for drug delivery, such as a long lifecycle in the blood stream, targeting to specific cells with ligands on the surface of the particles or cells and multiple drug targets [7, 11]. These designed nanocarriers must enter the cells and release the drugs to the target cells to execute their missions. Most nanoparticles enter the target cells through endocytosis [12]. The main endocytosis pathways are either clathrin dependent or clathrin independent [12]. The clathrin independent pathways include the micropinocytosis, caveolin dependent and caveolin independent (Arf-6, Flotillin, Cdc42 and RhoA- dependent) pathways [12]. After crossing the cell membrane, the nanoparticles are sequestered in vesicles. These vesicles are transported to early endosomes (EEs) and late endosomes (LEs) and finally fuse with lysosomes [12, 13]. Most developed nanoparticles, including PLGA-based nanoparticles, micelles, chitosan and microgels, have been shown to traffic in this classic endocytosis pathway. Modification of nanoparticles with various ligands, such as histidine, detergent and cell membrane penetrating peptides, enhances the nanoparticles’ escape from the endo-lysosome system [14-16]. The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy, which is responsible for the transport of various macromolecules between different organelles in the cells [17-19]. The crosstalk between endocytosis, exocytosis and autophagy makes these networks much more complex. Other endocytotic pathways exist in addition to the EEs-LEs-lysosome pathway. Recycling of the endosome system is responsible for delivering protein receptors back to the cell membrane [12]. The exocytosis pathway is responsible for the transportation of mature proteins from the Golgi body to the cell membrane or for secreting the proteins out of the cells [19]. Autophagy is a cellular process that engulfs cellular components, such as cytoplasm, damaged mitochondrial, aggregated proteins and invading pathogens [18]. Moreover, there are multiple pathways that exist in endocytosis, recycling endocytosis, exocytosis and autophagy. However, most studies focus on the intracellular trafficking and, accordingly, the design of nanocarriers is limited to the EEs-LEs-lysosome pathway. The trafficking pathways such as recycling endosomes, exocytosis and autophagy of the nanoparticles in the cells are poorly investigated and
unknown. The Rab protein family consists of GTPases/GTP- binding proteins that are evolutionary conserved from yeast to humans. Rab proteins have various cellular functions, including protein trafficking, transmembrane signal transduction and the fusion of vesicles between different organelles [20]. More than 70 human Rab proteins have been identified, and the functions of 30 Rab proteins have been identified [21]. Most of these Rab proteins are involved in the trafficking of vesicles in and across endocytosis, recycling endocytosis and exocytosis pathways [22]. Rab5 and Rab7 have been well studied and have become the most important organelle identity markers of EEs and LEs in the endocytic pathway, respectively [22]. To investigate in detail, the trafficking pathways of protein nanocapsules, we used these Rab proteins as vesicle markers to track the intracellular travel pathway of nanoparticles. The crosstalk between the endocytosis and exocytosis pathway with autophagy has also been extensively investigated, through the manipulation of the exocytosis and autophagy pathways with inhibitors such as 3-MA (autophagy inhibitor) and CQ (lysosome inhibitor), which block the degradation of the protein nanocapsules. The effects of the protein nanocapsules may extensively improve when combined with drugs that can block the autophagy and exocytosis pathways.
Materials and methods Materials Bovine serum albumin, dimethyl sulphoxide (DMSO), Chloroquine (CQ), and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-acryloxysuccinimide (NAS), acrylamide (AAm), N, N-methylene bisacrylamide (BIS), ammonium persulphate (APS), N, N, N’, N’-tetramethylethylenediamine (TEMED) and Fluorescein isothiocyanate (FITC) were purchased from Aladdin Industrial Co. LTD. (Shanghai, China). N-(3-Aminopropyl) methacrylamide hydrochloride was purchased from Polymer Science, Inc. All other chemicals of the highest quality were commercially available and used as received. Lyso-Tracker Red was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, Clathrin, Caveolin were from Cell Signaling Technology. The antibody against LAMP1 was from Santa Cruz Biotechnologies, and the antibody against β-actin was obtained from Abmart.
Plasmid and transfection The EGFP-LC3 and DsRed-LC3 plasmids were created in our laboratory. The DsRed-Rab5, http://www.thno.org
2101
Theranostics 2016, Vol. 6, Issue 12 DsRed-Rab7 and Flag-Rubicon plasmids were purchased from Addgene. The KSHV Flag-vBcl-2 plasmid was kindly provided by Professor Beth Levine from the University of Texas Department of Medicine. The Rab1-37 genes in the T Vector were from Professor Jiahuai Han’s Lab and sub-cloned into EGFP-C1 and DsRed-C1, respectively. All plasmids were confirmed by automated DNA sequencing. Cells were transiently transfected with the plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.
FITC-label nBSA at 37 ℃ for 20 h. For lysosome detection, the cells were incubated with Lyso-Tracker Red for 1 h. Then, the cells were washed with PBS three times and fixed with 4% paraformaldehyde for 20 min. Cells were stained with DAPI for 15 min and washed three times with PBS. Confocal microscopy was performed with a FLUO-VIEW laser scanning confocal microscope (Olympus, FV1000, Olympus Optical, Japan) in sequential scanning mode using a 60~100×objective. The operation processes were similar to reference [23].
Synthesis of nBSA
Autophagy assays
The synthesis procedures were similar to reference [4]. First, 20 mg of BSA in 4 ml of pH 8, 50 mM sodium carbonate buffer was reacted with 0.5 mg N-acryloxysuccinimide in 50 ml DMSO for 2 h at room temperature. Then, the reaction solution was thoroughly dialysed against pH 7.0, 20 mM phosphate buffer. Using 10 mg acryloylated BSA solution at 1 mg/ml, radical polymerization from the surface of the acryloylated protein was initiated by adding APS dissolved in deoxygenated and deionized water and TEMED into the test tube. A specific amount of N-(3-Aminopropyl) methacrylamide (Apm), AAm and BIS (molar ratio BSA/ Apm/ AAm/ BIS/ Aps/ TEMED = 1 / 500 / 2500 / 300 / 300 / 1200) dissolved in deoxygenated and deionized water was added to the test tube over 60 min. The reaction was allowed to proceed for another 60 min in a nitrogen atmosphere. Finally, dialysis was used to remove monomers and initiators. The unmodified BSA was removed using ion exchange chromatography.
Cells were transfected with EGFP-LC3 under the indicated conditions and then fixed in 4% paraformaldehyde. The percentages of cells with fluorescent dots representing EGFP-LC3 translocation were counted by confocal microscopy as described previously [24-26]. LC3II protein level was detected using an anti-LC3 antibody.
Characterization of nBSA Ion exchange chromatography was performed with a DEAE Sephadex A-25. Gel electrophoresis results were quantified with ImageQuant LAS4000 after agarose gel electrophoresis in 0.7% agarose gels. TEM images of nanocapsules were obtained with a Philips EM120 TEM at 100000X. Before observation, nanocapsules were negatively stained using 1% pH 7.0 phosphotungstic acid (PTA) solution. Zeta potential and particle size distribution were measured with a Malvern particle sizer Nano-ZS.
Cell culture MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS).
Cellular uptake of nBSA FITC was used as a model fluorescent molecule and was formulated in nBSA. Non-transfected or DsRed-Rab1-38 were incubated with 1 mg/mL
Immunoblotting Immunoblotting analysis was performed as previously described [24, 27]. In brief, cell lysates were resolved on 12% SDS-PAGE and analyzed by immunoblotting using an LC3 antibody, followed by enhanced chemiluminescence (ECL) detection (Thermo Scientific).
Immunofluorescence assay Briefly, the cells were incubated with the following primary antibodies: EEA1, Clathrin, LC3, Arf-6, Flotillin, Cdc42, RhoA, P62, EEA1, and Caveolin. TRITC and FITC labeled secondary antibodies were used to detect the primary antibodies.
Statistical methodology All results are reported as the mean ± S.D of three independent experiments. Comparisons were performed using two-tailed paired Student’s t tests (* P
