Vps34

Recombinant human PDGF was purchased from PeproTech (100-14B, United States). pPERK, ATF6, IRE1α, GRP78, PDI, VPS34, Beclin1, ATG5, LC3II, GAPDH and secondary antibodies were obtained from Abcam (Abcam, United States), PDI was purchased from Cell Signaling Technology (Danvers, MA, United States), NLRP3, Pro-Caspase1, Caspase1, GSDMD, GSDMDP30, IL-1 were obtained from Proteintech (Proteintech, China). Goat anti-rabbit and anti-mouse IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, United States). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad (Hercules, CA, United States). H&E kit was purchased from Beyotime.

30 μg of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk-TBS-0.1% Tween 20 for 1 h at room temperature. Antibodies against ATG7, ATG5, CCNA2, CCND2, CCND3, CCNE1, P53, p-P53, P21, RAD50, RAD51, KU70, KU80, MRE11 (Cell Signaling Technology, Danvers, MA, USA), RAB9A, BECN1, ULK1, VPS34, TOMM20 (Abcam, Cambridge, MA, USA) and LC3 (Novus Biologicals, Danvers, CO, USA) were applied to probe the membranes, respectively. The membranes were then washed five times in TBST and incubated with HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit, Cell Signal Technology, USA) diluted 1:2,000 in TBST for 1 h. After 5 times washes, the membranes were developed using an ECL kit (Biological Industries, Kibbutz Beit-Haemek, Israel).

After extraction of total protein by RIPA buffer containing phosphatase inhibitors (Biotech Well, Shanghai, China), the concentration of proteins was measured by The BCA protein assay kit (Beyotime, Shanghai, China). Equivalent amounts of protein in each sample were separated by 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and followed by transferred onto a polyvinylidenedifluoride (PVDF) membrane (BIO-RAD, United States). Subsequently, these protein was probed with the respective primary antibodies at 4°C overnight, including USP13 (1:1,000, No. PA5-106761, Thermo Fisher Scientific, Massachusetts, United States), AMPK (1:1,000, No. 4150, Cell Signaling Technology, Beverly, MA, United States), LC3 (1:1,000, No. 4599, Cell Signaling Technology, Beverly, MA, United States), Beclin 1 (1:1,000, No. 3495, Cell Signaling Technology, Beverly, MA, United States), VPS34 (1:1,000, No. ab124905, Abcam, Cambridge, United Kingdom) and GAPDH (1:3,000, No. 5174, Cell Signaling Technology, Beverly, MA, United States) followed by addition of appropriate HRP-conjugated goat anti-rabbit IgG (1:5,000, No. S0001, Affinity Biosciences, Beijing, China) at 37°C for 1 h. GAPDH was used as an endogenous reference. The protein signal was analyzed by ECL assay kit (enhanced). Each trial was performed in triplicate.