Sa00001 2

Protein extracts from kidney tissues (renal cortex) and cells were obtained by using radio-immunoprecipitation assay (RIPA) buffer and phenylmethylsulfonyl fluoride (PMSF). Then, the protein concentration was determined using a BCA Protein Concentration Detection Kit (P0009; Beyotime, Shanghai, China). The protein lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% bovine serum albumin (BSA), the membranes were treated with primary antibodies against mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3; 14,702-1-AP), Bcl-2 (26,593-1-AP), Bax (50,599-2-Ig), Bcl-xl (10,783-1-AP), cleaved caspase-3 (AF1150), or β-actin (60,008-1-Ig) at 4°C overnight. Subsequently, the membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (SA00001-1 and SA00001-2; Beyotime) at 37°C for 40 min and visualized with the enhanced chemiluminescence (ECL) substrate. All primary antibodies were purchased from Proteintech except for cleaved caspase-3 (Beyotime). β-actin served as the internal control.

Cell protein were harvested using RIPA lysis buffer (Beyotime) supplemented with phenylmethanesulfonyl fluoride (Beyotime). The extracted proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, St. Louis, MO, USA). The membranes were incubated with primary antibodies including anti-FAM227A (1∶1000 dilution; Cat. #STJ193640, St John's Laboratory, London, UK), anti-AhR (1∶1000 dilution; Cat. #ET1703-11, HUABIO), anti-CYP1A1 (1∶1000 dilution; Cat. #13241-1-AP, Proteintech, Wuhan, China), or anti-GAPDH (1∶1000 dilution; Cat. #AF0006, Beyotime) as the internal control. HRP-conjugated anti-rabbit or anti-mouse antibodies (1∶2000 dilution; Cat. #SA00001-1 or SA00001-2, Beyotime) were then used. Signals were observed and imaged by using an ECL system (Thermo Fisher Scientific).