Goat anti mouse rabbit secondary antibody

Total proteins were extracted from mouse iWAT and pVAT using a Total Protein Extraction kit (Applygen, Beijing, China), and the protein concentrations were measured with a BCA protein assay reagent kit (Applygen, Beijing, China). Approximately 30 μg of protein was separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA) through a wet transfer (BIO-RAD, California, USA). The membranes were incubated with the primary antibodies (anti-UCP1, Proteintech, Wuhan, China; anti-PGC1α, Proteintech, Wuhan, China; anti-ZAG, Santa Cruz, Dallas, USA; anti-β-actin, CST, Danvers, MA, USA; anti-GAPDH, CST, Danvers, MA, USA) at a 1:100 to 1:1,000 dilution at 4°C overnight, followed by incubation with a goat anti-mouse/rabbit secondary antibody at a 1:5,000 dilution for 1 h (ZSGB-BIO, Beijing, China). The specific protein bands were visualized by enhanced chemiluminescence (Applygen, Beijing, China). The protein bands were quantified by Quantity One software (Version 4.6.9, BIO-RAD, California, USA). The expression levels of target proteins in iWAT were normalized to that of β-actin, and the expression levels in pVAT were normalized to that of GAPDH.

OSCC cell lines (HSC-3, HSC-4) were received from the Japanese Collection of Research Bioresources (JCRB) Cell Bank and the Cal-27 and UM1 cell lines were kindly provided by Dr. J. S. Gutkind (National Institute of Dental and Craniofacial Research). The cells were routinely cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies) at 37 °C in 5% CO₂ incubator. Silmitasertib, bafilomycin A1 (BAF1), Z-VAD-FMK were purchased from Selleck. Cisplatin (DDP) was purchased from Sigma-Aldrich. The Cell counting kit-8(CCK8) was purchased from Dojingdo (Kumamoto, Japan). The Annexin V-FITC/PI apoptosis double staining kit was obtained from Keygen Biotech Co. Ltd (Nanjing, China). AF488 Dextran was purchased from Thermo Fisher Scientific. Lyso-tracker Red, Mito-Tracker Red, ER-Tracker Red and Hoechst 33342 were obtained from Beyotime (Shanghai, China). The primary antibodies anti-cleaved Caspase 3, anti-cleaved Caspase 8, anti-ERK, anti-p-ERK, anti-p38, anti-JNKs, anti-p-JNKs, anti-β-actin, and anti-Ki67 were obtained from Abcam (1:1,000). Goat anti-mouse/rabbit secondary antibody was purchased from ZSGB-BIO.