Sc 134882

Cells were washed once in PBS and lysed in RIPA lysis buffer (P0013B; Beyotime, Shanghai, China) at 4°C. Proteins were denatured by boiling. Protein concentrations were determined using the Enhanced BCA Protein Assay kit (P0010S; Beyotime). Protein samples were separated in a 12% SDS-polyacrylamide gel and transferred to PVDF membranes (Immobilon-P membranes, Millipore, USA). Membranes were blocked with Tris-buffered saline/Tween 20 (TBST, 0.1% Tween 20) containing 5% bovine serum albumin (BSA) for 1 h and then incubated with the appropriate primary antibody overnight at 4°C. After three 10-min washes with TBST, membranes were incubated for 1 h at 24°C with the appropriate horseradish peroxidase-conjugated secondary antibody. After extensive washing, immunoreactive bands were detected by the BeyoECL Plus reagent (P0018; Beyotime) using a Photo-Image System (Molecular Dynamics, Sunnyvale, CA, USA). Immunoblotting was performed with the following primary antibodies: phosphorylated NRF2 Ser40 (pNRF2 Ser40) (ab76026; Abcam), NRF2 (ab62352; Abcam), GCLC (ab53179; Abcam), GGT1 (ab175384; Abcam), NQO1 (ab34173; Abcam), HO1 (ab13243; Abcam), Retinoblastoma (RB) (ab24; Abcam), p53 (ab1101; Abcam), p21 (ab109520; Abcam), p16 (ab51243; Abcam), CRIF1 (sc-134882; Santa Cruz), PKC-δ (sc-213-G; Santa Cruz), and β-actin (sc-8432; Santa Cruz).