Anti p akt anti alix

The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).

The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).