Bca protein assay reagent kit

Liver proteins were collected using RIPA lysis buffer (C1053, Applygen Technologies, Beijing, China) containing protease/phosphatase inhibitor cocktail (GRF101/102, EpiZyme, Shanghai, China). Protein concentrations were determined using the BCA protein assay reagent kit (P1511, Applygen Technologies, Beijing, China). Protein samples were separated by SDS-PAGE gels and transferred onto a polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). After blocked with 5% nonfat dry milk (PS112L, Epizyme, Shanghai, China) overnight at 4 °C, the membranes were incubated successively with specific primary and secondary antibodies. The protein blots were visualized with Omni-ECL™ Femto Light Chemiluminescence Kit (SQ201, Epizyme, Shanghai, China) and automatic exposure system (Image Quant LAS500, GE, Fairfield, CT, USA).

Mouse lung tissues or cells were lysed with RIPA Lysis Extraction Buffer (Beyotime Technology) along with protease inhibitor cocktail (Selleck). The total protein concentration was determined by a BCA protein assay reagent kit (Applygen Technologies Inc.) according to the manufacturer’s protocol. Total protein (20 µg) was loaded and separated by 10% SDS‒PAGE and then transferred to a PVDF membrane. Membranes were blocked with 5% skim milk in TBST buffer for 1 h and then incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: anti-ZNF451 (Proteintech), anti-α-SMA (BOSTER), anti-Col1 (Abcam), and anti-GAPDH (ZSGB BIO). The signaling was visualized using a ChemiDocTM XRS + with Image LabTM Software (Bio-Rad, Hercules, California, USA) with an ECL kit (Tanon).

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.