Complete protease and phosphatase inhibitor cocktail

M-PER (Mammalian Protein Extraction Reagent; Thermo, USA) and a complete protease and phosphatase inhibitor cocktail (Roche, Switzerland) were added to culture flasks. The cells were then scraped off and shifted to centrifuge tubes. The supernatant whole-cell extracts were obtained, and the BCA™ Protein Assay Kit (Pierce, USA) was used to determine the protein concentration. The weights of the protein samples were adjusted to 35 µg, and the proteins were then denatured at 100°C and subjected to electrophoresis on 8% SDS-PAGE. The proteins were transferred from gels to nitrocellulose membranes (Invitrogen, USA), and the membranes were blocked with 5% nonfat dry milk. The proteins were incubated with anti-NPG-12 antibodies (1 µg/ml) and anti-NSP antibodies (1 µg/ml) overnight at 4°C, and a commercially available anti-fgl2 antibody (2 µg/ml, Abnova, Taiwan) was used as a positive control. HRP-conjugated (Sigma, USA) antibodies were used as secondary antibodies. The specific bands were visualized using the ECL reagent (Pierce, USA) and recorded by the Molecular Imager ChemiDoc XRS System (Bio-Rad, USA) [34] (link).

For MST2 immunoprecipitation, cells were treated as indicated and washed with ice‐cold PBS prior to lysis. Cells were lysed in 1% NP‐40 lysis buffer (150 mM NaCl, 20 mM HEPES, 0.5 mM EDTA) containing complete protease and phosphatase inhibitor cocktail (Roche). Total cell extracts were incubated for 3 h with 20 μl protein A Dynabeads (Invitrogen) and 2 μg of MST2 antibody (ab52641) at 4°C. Total cell extracts (corresponding to 10% of the immunoprecipitate) and immunoprecipitates were resolved in 4–12% Bis–Tris Nu‐PAGE gels (Invitrogen) and transferred onto PVDF membrane (Millipore) before immunoblotting with the appropriate antibodies overnight at 4°C. Primary antibody detection was achieved with peroxide‐conjugated anti‐rabbit or anti‐mouse antibodies (Jackson Immunoresearch) and exposure to X‐Ray film (Kodak). To quantify the bands obtained with western blot analysis, we used ImageJ software (NIH). All bands were normalised against the loading controls. For detection of H2BS14, the phospho‐histone H2B (Ser14; Millipore, 07‐191) was used unless stated otherwise.

Human mammary tissue-derived cell lines were procured from American Tissue Culture Collection (ATCC, VA, USA). MCF10A cells (cat. # CRL-10317, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium supplemented with 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 20 ng/mL EGF, 10 μg/mL insulin, and 5% horse serum (Fisher Scientific). MCF7 (cat. # HTB-22, ATCC) and MDA-MB-231 (cat. # HTB-26, ATCC) cells were cultured in high-glucose DMEM supplemented with 10% (v/v) fetal bovine serum (Fisher Scientific) and 10 μg/mL insulin (MCF7 only). All cells were cultured in 5% CO₂ at 37 °C. Replicate cell cultures were harvested at sub-confluence by scraping in 1x phosphate buffered saline. Cells were centrifuged at 7000 × g for 1 min, snap-frozen in liquid nitrogen and stored at −80 ^oC until use. Cell lysis was performed by resuspension and Dounce homogenization in buffer containing 10 mM Tris-HCl, 250 mM sucrose, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 0.1% (v/v) dodecyl-β-D-maltopyranoside (DDM) and 1× Complete Protease and Phosphatase Inhibitor Cocktail (Roche). The homogenates were treated with Turbonuclease (100 units/mL) (Accelagen) for 30 min at 4 ^oC, clarified by centrifugation at 18,000 × g for 20 min at 4 °C, quantified by Bradford assay (Bio-Rad) and adjusted to 6.0 mg protein/mL prior to fractionation.

For western blot analysis of primary adipocytes, cells were washed twice with KRBH medium without BSA and subsequently lysed in lysis buffer containing 50 mM Tris/HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.27 M sucrose, 1% NP-40, and complete protease- and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Lysates were centrifuged for 10 min at 13000xg, and protein concentrations were determined using the Bradford method. Samples were subjected to polyacrylamide gel electrophoresis and electro-transfer to nitrocellulose membranes. Membranes were blocked with non-fat dry milk [5% (w/v)] and probed (overnight, 4°C) with the indicated antibodies. Detection was performed using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagent. The signal was visualized using a BioRad Image camera (Biorad, Hercules, United States). All data were normalized using HSP90 as a loading control.

The whole-cell lysates were prepared with extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.5% Nonidet P-40) supplemented with complete protease and phosphatase inhibitor cocktail (Roche). The procedures for standard western blotting were performed according to the manufacturer’s guidance. The antibodies specific for TRAF3 (Cat#: 4729, 1:1000 dilution), P100 (Cat#, 4882, 1:1000 dilution), P52 (Cat#, 4882, 1:1000 dilution), IKβα (Cat#, 2859, 1:1000 dilution), and β-actin (Cat#, 4970, 1:1000 dilution) were purchased from Cell Signaling Technologies, USA. The uncropped and unprocessed scans of Fig. 4c are shown in Supplementary Fig. 16.

Western blotting was performed as described previously [30 (link)]. In short, following incubations, cells were washed with KRH medium without BSA and subsequently lysed in a buffer containing 50 mM Tris/HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 0.27 M sucrose, 1% NP-40, and complete protease-and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) (one tablet each/10 ml buffer). Lysates were centrifuged for 10 min at 13000xg and protein concentrations were determined using the Bradford method. Samples were subjected to polyacrylamide gel electrophoresis and electro-transfer to nitrocellulose membranes. Membranes were blocked and probed with the indicated antibodies. Detection was performed using horseradish peroxidase conjugated secondary antibodies and enhanced chemiluminescence reagent. The signal was visualized using a BioRad Image camera (Biorad, Hercules, USA).