Enhanced chemiluminescence system kit

Anti-AP2M1, anti-cytochrome C and anti-cleaved caspase-3 antibodies were obtained from Abcam (Shanghai, China). Anti-Beclin 1, -p62, -LC3, bcl-2, -bax, -cleaved-caspase3 and GAPDH antibodies were obtained from ProteinTech (Shanghai, China). Total proteins were extracted using RIPA-lysis buffer supplemented with 10 mM PMSF (Beyotime Institute of Biotechnology, Shanghai, China). A bicinchoninic acid assay was used to quantify protein concentrations. A total of 40 μg protein was loaded on a 10% SDS-gel, resolved using SDS-PAGE and transferred to polyvinylidene fluoride membranes. Then, the membranes were blocked with 5% nonfat milk in Tris-buffered saline and 0.1% Tween 20 (TBST), and incubated with primary antibodies at 4 °C overnight. The following day, the membranes were washed 5 times with TBST, the membranes were then incubated with secondary antibodies for 2 h. An enhanced chemiluminescence system kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to visualize the signals. Densitometry analysis was performed using ImageJ.

Western blot analysis was performed as described [30 (link)]. For CAPN5 or scFvs overexpression experiments and scFv protein treatment experiments, Cells were transfected with plasmids for 60 h, or treated with scFvs at 10 μg/ml in DMEM with 0.1% FBS for 24 hours, after transfections and treatments, cells were collected and lysed. The total cell proteins were separated by SDS-PAGE and transferred to PVDF membrane, the membranes were incubated with rabbit anti-activated caspase 3, caspase 9 antibodies, goat anti-CAPN5, monoclonal anti-IL1alpha, anti-His, or anti-C-myc antibodies in 5% milk which diluted in PBS at 4°Covernight respectively, after washing with 0.1%TBST, the membrane incubated with donkey anti-mouse/goat IgG HRP secondary antibodies for 1 hour at room temperature.
For H₂O₂ exposure experiments, cells were pre-treated with 10 μM H2O2 for 2, 6, 12, 24 hours, then the cells were collected and lysed for immunoblot analysis as above. Bands were visualized with an enhanced chemiluminescence system kit (Beyotime). Signals were detected and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).