Anti nur77 antibody

Cell lysates were extracted using radioimmunoprecipitation assay (RIPA) buffer containing (in mM, pH 7.5) 20 Tris-HCl, 150 NaCl, 1 EGTA, 1 % NP-40 with protease and phosphatase inhibitor and protein concentration was quantified using bicinchoninic acid (BCA) reagents (ThermoFisher Scientific). 10–12 % SDS-acrylamide gel were used for electrophoresis and transfer into nitrocellulose membrane and then blocked with 3 % BSA in TBST. Anti-Nur77 antibody was purchased from Novus Biologicals (USA, Cat#： NB100-56745), anti-GCH1 antibody was purchased from Abcam (Cat# ab307507), anti-SOD1 antibody was purchased from Sigma (Cat#:SAB5701040), and anti-GAPDH antibody was purchased from Santa Cruz (Cat#: sc-365062). Antibodies were diluted with 3 % BSA in TBST and incubated at 4 °C for overnight. Next day, blots were washed with TBST for 3 times and then incubated with IRDye® 800 CW goat anti-rabbit or IRDye 680RD goat anti-mouse secondary antibody (1:10,000, LI-COR) for 1 h at room temperature. Band intensity was quantified with Image J (version 1.32j, NIH) from three to five independent experiments.

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco/Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco's phosphate-buffered saline (DPBS) was purchased from Invitrogen/Thermo Fisher Scientific. In addition, 3-methyladenine (3-MA) and rapamycin (RAPA) were purchased from Selleck Chemicals (Shanghai, China), and leptomycin B (LMB) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). The anti-Nur77 antibody was purchased from Novus Biologicals, and anti-LC3 (cat. nos. 12741 and 2775) and anti-GAPDH antibodies (cat. no. 5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Beclin-1 (cat. no. ab210498) and anti-Bcl2 (cat. no. ab692) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-rabbit and mouse IgG (ZB2301, ZB2305, 1:2,000) were purchased from Zhong Shan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).