Goat anti mouse igg

After treatments, the cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (Beyotime, Beijing, China) with protease inhibitors, sonicated for 15 seconds and centrifuged at 12,000 g for 15 minutes at 4 °C. The protein concentrations were determined by a BCA protein assay kit (Beyotime, Beijing, China), and then the protein samples were mixed with loading buffer and heated at 100 °C for 10 minutes. Equal amounts of protein samples were loaded per lane, separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore), which were then blocked with 5% non-fat milk for 3 hours. The PVDF membranes were then probed with different antibodies: rabbit anti-LAMP2 (1:1500, Abcam), rabbit anti-Beclin1 (1:1000, CST), rabbit anti-LC3 (1:1000, CST), rabbit anti-cleaved-caspase3 (1:1000, CST) and mouse anti-β-actin (1:5000, Sungene Biotech). After the primary antibody binding, the membranes were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit (1:8000, MultiSciences, GAR007, China) or goat anti-mouse IgG (1:8000, MultiSciences, GAM007, China) at room temperature for 1 hour. The reactive proteins were detected by an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA), the band intensities were quantified with Quantity One software, and the results were normalized to β-actin.

Cells were rinsed twice with PBS, sonicated for 15 seconds in 500 μl of RIPA lysis buffer (Beyotime, China), and centrifuged at 14,000g for 5 minutes. Protein concentration was then determined by bicinchoninic acid protein assay (Beyotime, Beijing, China). The sample loading buffer was added to the protein sample and heated at 100°C for 10 minutes. The proteins were separated by electrophoresis in 10% tris-glycine polyacrylamide gradient gels. The separated proteins were then transferred onto PVDF membrane (Millipore), blocked with 5% skimmed milk for 2 hours, and incubated with rabbit monoclonal anti-CD36 antibody (1:800, Abcam, ab133625, UK), or rabbit monoclonal cleaved-caspase3 antibody (1:1000, Cell Signaling Technology, 9664S, USA) overnight at 4°C. After washing and incubation with horseradish peroxidase (HRP)-labeled Goat anti-rabbit (1:8,000, MultiSciences, GAR007, China) or Goat anti-mouse IgG (1:8,000, MultiSciences, GAM007, China) at room temperature for 1.5 hours, the membranes were probed with chemiluminescence reagents using a commercially available kit (Pierce Biotechnology, USA). Protein expression was detected using a chemiluminescent staining reagent kit to visualize the signals, followed by exposure to x-ray films. Band intensities were quantified with Quantity One software, and calculated as the optical density×area of the band.