Ab99421

Western blot analysis was designed to assess the expression of autophagy and pyroptoysis‐related proteins. Target protein concentrations were determined using a BCA protein assay kit (Pierce). Equal amounts of proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were than immersed in Tris‐buffered saline Tween (TBST) containing skimmed milk and left for 1 hour at room temperature. Thereafter, the membrane was added with a suitable concentration of primary antibody NLRP3 (ab214185, Abcam), USP13 (ab99421, Abcam), NF‐κB (abs131170, absin), Beclin 1 (3738, CST), GSDMD (93709s, CST), GSDMD‐N (ab215203, Abcam), Cleaved IL‐1β (AF4006, Affinity), ASC (67824, CST), LC3 I and LC3 II (12741, CST) and GAPDH (AF7021, Affinity) at 4°C, and then washed three times with TBST. After that, the membrane was incubated with the horseradish peroxidase‐labelled secondary antibody Goat Anti‐Mouse IgG (H + L) HRP (1:5000, No. S0002, Affinity) or Goat Anti‐Rabbit IgG (H + L) HRP (1:5000, No. S0001, Affinity) at room temperature for 2 hours. Afterwards, the blot was detected using enhanced chemiluminescence. The gel analysis system scanned each strip protein, and the grey value of the strip was measured by image analysis software (Image J).

Antibodies to USP13 (ab99421), NF-kB p65 (ab16502), NF-kB phosph-p65 (ab86299), pan-AKT (ab8805), phosph-AKT (Ser473) (ab8932), phosph-AKT (Thr308) (ab8933), PTEN (ab170941) and GAPDH (ab181602) (all from abcam) were used according to the manufacturers’ protocols. The western blotting analysis was performed as described before. Briefly, equal amounts of protein extracts were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20) with 5% (w/v) non-fat dry milk and were then incubated with primary antibodies in TBS-T at 4 °C overnight. After three washes with TBS-T for 15 min each, the membranes were incubated with the appropriate HRP-labeled secondary antibodies for 1 h at 37 °C. The immunobands were visualized using the ECL reagents (Transgen Biotechnology, Beijing, China) on a MicroChemi Chemiluminescent Imaging System (DNR Bio-Imaging Systems, Mahale HaHamisha, Jerusalem, Israel). The densitometric values for each band were calculated by Image J 1.46r software (Wayne Rasband, National institutes of Health, Bethesda, MA, USA), and the statistical analysis were conducted based on the ratios of target protein/GAPDH.

Immunohistochemistry analysis was performed as previously described (Dou et al., 2019b (link)). The primary USP13 antibody (ab99421) and Ki-67 antibody (ab15580) were obtained from Abcam (Cambridge, MA, United States). Not detected and low staining of USP13 were recognized as negative expression, and medium and high staining of USP13 were defined as positive expression. The percentage of positive staining cells was calculated for quantifying Ki-67 staining.