Ecl plus assay kit

Cardiac tissues and cardiomyocytes extracts were used for Western blot analysis. The equal amount of protein samples obtained from tissues or cells were separated by SDS-PAGE electrophoresis and then transferred to PDVF membrane. The membranes were incubated with the following primary antibodies: USP14 (1191S, CST, USA), Beclin1 (3738, CST), LC3 (12741, CST), NCOA4 (ab86707, Abcam) and FTH1 (3998, CST) and transferrin receptor 1 (TfR1; ab1086, Abcam). Secondary antibodies were incubated and then visualized with the ECL Plus assay Kit (K002, Affinity, USA).

The steps of protein extraction and Western blot analysis are referred to the previous literature.¹⁵ In brief, the extracted bladder protein tissue was loaded into a 10% SDS‐polyacrylamide gel and transferred into a PVDF membrane after electrophoresis. After incubating with primary and secondary antibodies and washing (NLRP3, ASC, Pro‐caspase‐1, Caspsae‐1 p20, GSDMD, GSDMD‐N and Cleave‐IL‐1β, Affinity; Goat Anti‐Rabbit IgG (H + L) HRP, Affinity), the ECL Plus assay Kit (Affinity, K002) was used for colour rendering. The gel analysis system scanned each strip protein, and the grey value of the strip was measured by image analysis software (Image J).