Nuclear and cytoplasm protein extraction kit

Total cell extracts were prepared in 1× sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio, China). Cell proteins were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with GAPDH (1:1000, BM3876, Bosterbio, USA), p-PERK, p-IRE1, ATF4, P16 (1:500, bs-3330R, bs-16698R, bs-1531R, bs-20656R, Bioss, China), H3, NRF2 (1:500, D153567, D121053, Sangon Biotech, China), and TNF-α, IL-6, P21, MT1, MT2, HRD1, VCP, P65, p-P65, IKK (1:200, sc-52746, sc-32296, sc-136020, sc-13180, sc-13177, sc-293484, sc-136273, sc-514451, sc-166748, sc-7606, Santa Cruz, USA). Secondary anti-rabbit, anti-mouse antibodies (1:1000, BM2004, BA1001, Bosterbio, USA) conjugated with horseradish peroxidase were used. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence western blotting substrate (Thermo Scientific). Results were analyzed by Tanon-410 automatic gel imaging system (Shanghai Tianneng Corporation, China).

Total cell extracts were prepared in 1 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio). Cell proteins were resolved by SDS‐PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were incubated with primary antibodies overnight at 4°C and appropriate HRP‐secondary antibodies for 1 hr at room temperature. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence Western blotting substrate (Thermo Scientific) (Xiong et al., 2016).