Protease inhibitor cocktail

The pancreatic tissue was collected and ground in liquid nitrogen. The samples were harvested into universal protein extraction lysis buffer (Cat. No. PP1801, Bioteke, Beijing, PR China) containing a protease inhibitor cocktail (Cat. No. 04693116001, Roche, Basel, Switzerland). After the protein concentration was determined using the BCA method (Bioteke, Beijing, China), 20–50 μg of total cell protein was separated via SDS-PAGE, transferred to Polyvinylidenefluoride (PVDF) membranes (Cat. No. IPVH00010, Millipore, Massachusetts, USA), and incubated with the appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) western blot detection system (Millipore). The optical densities of the bands were measured using Image Pro plus 6.0 (IPP 6.0, Media Cybernetics, Silver Springs, MD, USA) software. The relative expression levels were calculated as the ratio of the optical density of the target protein to GAPDH.

After cells were transfected with the designated plasmids, cells were harvested into universal protein extraction lysis buffer (Cat. No. PP1801, Bioteke, Beijing, PR China) containing protease inhibitor cocktail (Cat. No. 04693116001, Roche, Basel, Switzerland). Extracted proteins were incubated 1–3 μg target antibody or IgG as negative control overnight, and followed with protein A+G agarose beads (Cat. No. P2012, Beyotime, Jiangsu, PR China) for 2 h. Precipitated proteins were subjected to SDS-PAGE, transferred to PVDF membrane (Cat. No. IPVH00010, Millipore, Massachusetts, USA), and detected with specific appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) Western blot detection system (Millipore).