Specific primary antibodies against cleaved caspase 3

Total proteins were extracted using RIPA lysis buffer. Protein concentration was determined using a BCA Protein Assay Kit (Tiangen, Beijing, China). Equal amount of protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking in 3% BSA, the membranes were incubated with specific primary antibodies against cleaved caspase 3, total caspase 3, cleaved PARP, total PARP, Bcl-2, and GAPDH (all from Abcam, MA, USA) at 4 °C overnight. After washing TBST for 3 times, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA), and the band intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).

Following the lysis of isolated islet cells with RIPA buffer, the protein content was determined using the method described by Lowry et al.^{57 (link)}. Subsequently, equivalent quantities of proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride membranes. To block non-specific binding sites on the membranes, 50 g/l nonfat milk in TBST (20 mMTris-Hcl, pH 7.5, 150 mMNaCl, 1 g/l Tween20) washing buffer was applied for 2 h at room temperature. The membranes were then incubated overnight at 4 °C with specific primary antibodies against cleaved-caspase 3 (1:5000, Abcam, Cambridge, United Kingdom) and caspase-9 (1:2000, Abcam, Cambridge, United Kingdom). After necessary washes, the membranes were incubated with peroxidase-labeled secondary antibodies at a dilution of 1:20,000 (Abcam, Cambridge, United Kingdom) at room temperature for 1 h. Immunoreactivity was detected using the ECL detection kit (Bio Rad Laboratories India Pvt Ltd) through chemiluminescence. Gel-Pro Analyzer 6.0 application was employed for densitometry to measure band intensity in relation to the β-actin bands (Media Cybernetics, Silver Spring, MD, USA).