Anti mouse igg secondary antibody

S. coelicolor cells grown to OD₆₀₀ of 0.4–0.5 in YEME were sonicated in lysis buffer (20 mM Tris-HCl, pH 7.9, 10% glycerol, 5 mM EDTA, 0.1 mM DTT, 10 mM MgCl₂,1 mM PMSF and 0.15 M NaCl). Protein concentration in crude cell extracts was determined by Bradford reagent solution (Bio-Rad) using BSA as a standard. Either purified Zur or cell extracts containing 5 μg proteins were electrophoresed on 13% SDS–PAGE. Polyclonal mouse antibody against Zur (prepared in our lab) and the anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, SC-2005) were used at 1:5,000, 1:3,000 dilution ratio, respectively, for immunodetection (Fusion Solo; Vilber Lourmat). A representative full-sized immunoblot photo is shown in Supplementary Fig. 13. All experimental protocols that involve animals were approved by and done in accordance with the guidelines by Seoul National University Institutional Animal Care and Use Committees (SNUIACUC).

Antibodies against UCP1 (uncoupling protein 1; 1:1000; #14670), Sirt1 (sirtuin-1; 1:1000; #3931), PPAR-γ (peroxisome proliferator-activated receptor gamma; 1:1000; #2435), PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha; 1:1000; #2178) and a secondary antibody (anti-rabbit IgG, HtL; 1:5000) were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against β-actin (1:20,000; NB600-501) and an anti-mouse IgG secondary antibody (1:20,000; sc-2005) were purchased from Santa Cruz Biotechnology (Dallas, TE, USA).

To isolate proteins, cells were lysed with RIPA buffer (Sigma, cat no. R0278), supplemented with PMSF and protease inhibitors (Roche, cat no. 11836153001). Protein concentration was estimated using the BCA assay (Thermo Scientific). Protein was separated using polyacrylamide gel electrophoresis (4–15%) and transferred to a PVDF membrane (Bio‐Rad Inc.). The membrane was blocked in 5% milk and incubated with anti‐SREBP antibody (1:250; Santa Cruz, cat no. sc‐365513) overnight at 4 degrees followed by anti‐mouse IgG secondary antibody (cat no. A11008) in TBS‐T for 1 hr. The signal was observed using SuperSignal West Dura Extended Duration substrate (Thermo Scientific) and images captured on the Bio‐Rad ChemiDoc imager and analyzed using Fiji (Schindelin et al., 2012).