K1015

Cells were washed with cold phosphate-buffered saline (PBS) and lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl and 0.5% NP-40) supplemented with protease inhibitors (K1019, APExBIO, Houston, TX, USA) and phosphatase inhibitors (K1015, APExBIO, Houston, USA). The cell lysates were clarified by centrifugation at 13,200 r.p.m. at 4 °C for 10 min. The protein concentrations of lysates were measured using Nanodrop with Bio-Rad protein assay reagent. Equal amounts of whole-cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, 2000–5000 μg lysates were incubated with agarose-conjugated primary antibodies for 3–4 h at 4 °C or with the indicated primary antibody (3 to 5 μg) overnight followed by 1 h incubation with Protein A Sepharose beads. Immunoprecipitants were washed three times with NETN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40) and then resolved by SDS-PAGE. Anti-HA agarose beads (A2095) and anti-Flag agarose beads (A2220) were purchased from Sigma Aldrich. Anti-Myc agarose beads (658502) were purchased from BioLegend. Anti-PRMT5 antibody conjugated to agarose (sc-376937) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Cells were collected and lysed with RIPA buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% NonidetP-40, 0.5% sodium deoxycholate, 1% protease inhibitor (HY-K0010, MCE, Monmouth Junction, USA), and 1% phosphatase inhibitor (K1015, APExBIO, Houston, USA) mixture. The RIPA buffer used in this study does not contain SDS. Lysates were prepared, and protein concentration was measured using a Bio-Rad Protein Assay (Bio-Rad, Hercules, USA). Samples were stored at -20°C until use.
For Co-IP, cells were collected and lysed with NP-40 buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% NP-40, and 1% protease and phosphatase inhibitor. Then, 20μl protein A/G agarose was added to the lysates to perform prewash for 2 h, and the appropriate amount of IgG or indicated antibodies was added to the supernatant at 4°C overnight according to the instructions. The following day, the target protein in the lysates was captured by adding protein A/G agarose. The final product was washed five times with NP-40 buffer containing 1M NaCl and stored at -20°C until use. All total protein and Co-IP samples underwent sodium dodecyl sulfate-polyacrylamide electrophoresis, transfer to nitrocellulose membranes, and detection with the indicated antibodies.

The 100‐mg gonadal adipose tissue was lysed in 1‐mL radioimmunoprecipitation assay (RIPA) buffer with phenylmethylsulfonyl fluoride (PMSF, P0100; Solarbio Co., Ltd.) and phosphatase inhibitor (K1015; Apexbio) for 30 min. The volume ratio of RIPA buffer, PMSF, and phosphatase inhibitor is 100:1:1. The lytic product was centrifuged at 4°C with 14,000 g for 15 min. Equal amounts of proteins (30 μg/well) were loaded into SDS‐PAGE, separated by electrophoresis at 120 V, and transferred on PVDF membranes (Millipore) at 220 mA (Bio‐Rad). After that, the membranes were sealed at room temperature with 5% nonfatty milk for 4 h and incubated overnight at 4°C in an appropriate amount of diluted primary antibodies (anti‐Nrf2 1:500, anti‐phospho‐Nrf2 1:500, anti‐UCP1 1:1000, anti‐PGC1α 1:1000, and β‐actin 1:500) according to the instructions. Then, membranes were washed twice with TBST buffer for 15 min each time, after incubating at room temperature in the second antibody (1:2000) for 2 h, membranes were washed twice again with TBST buffer, each for 15 min. The signals were finally detected on Molecular Immer ChemiDoc XRS System (Bio‐Rad) with an ECL detection kit (Beyotime). The band intensity ratio of the target protein to β‐actin indicates the relative protein expression level.