Beclin 1 3495 s

Primary antibodies recognizing LC3B (#3868), P62 (#8025), ATG5 (#4445), ATG7 (#4445), BCL-2 (#15071), BAX (#5023), cleaved-caspase 9 (#7237), beclin-1 (#3495 S) and HO-1 (#43966) as well as the secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody NRF2 (sc-365,949) was purchased from Santa Cruz (Dallas, TX, USA). Beclin-1-shRNA and negative control shRNA (NC-shRNA) vectors were purchased from GenePharma Co., Ltd. (Shanghai, China). Sulforaphane (HY-13755), chloroquine (HY-17589), Z-VAD-FMK (HY-16658 B) and NRF2 inhibitor ML385 (HY-100523) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Whole cell lysates were prepared with RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (B14012, Bimake, USA). Protein concentrations were determined using the BCA protein assay (23229, Thermo Fisher Scientific, USA). Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to the appropriate PVDF membranes (0.22 µm: ISEQ00010; 0.45 µm: IPVH00010, Millipore, USA). The membranes were blocked with 5% nonfat dry milk at room temperature for 1 h and then incubated overnight at 4 °C with primary antibodies against Caveolin-1(16447-1-AP, Proteintech, USA), LC3 A/B (4108S, Cell Signaling Technology, USA), Beclin-1 (3495S, Cell Signaling Technology, USA), IRGM (AP11128b, Abgent, USA), p62 (sc-28359, Santa, USA) and β-actin (sc-58673, Santa, USA). Protein bands were visualized through Immobilon Western chemiluminescent reagents (WBKLS0500, Millipore, USA).