Complete protease inhibitor cocktail

Mouse hippocampi were homogenized in chilled TBS supplemented with a protease- and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations of the homogenates were determined by BCA assay (CWBIO). Samples were electrophoretically separated on 10 or 12% SDS–PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h and probed with primary antibodies (Anti-UCP-1 antibody ab10983) at 4 °C overnight. The membranes were subsequently developed with the corresponding horseradish peroxidase-conjugated secondary antibody and ECL kit (Thermo Fisher Scientific). Densitometry quantification of the protein bands was analyzed using IMAGE-PRO PLUS 6.0 imaging software.
The mixture of proteins was extracted from scapular brown adipose tissues in RIPA buffer (0.5% NP40, 0.1% sodium doxycycline, 150 mM NaCl, 50 mM Tris.HCl [pH 7.4]) containing a complete protease inhibitor cocktail (Abcam). PVDF membranes were probed simultaneously with primary antibodies of UCP1 (Abcam) and β-tubulin (Abcam). The protein bands were visualized with ECL chemiluminescence reagents (Thermo Fisher Scientific) and quantified using IMAGE-PRO PLUS 6.0 imaging software.

Total proteins were extracted from cells using lysis buffer supplemented with the complete protease inhibitor cocktail (Abcam). The following primary antibodies were used for Western blot analysis: anti‐PTBP1 (ab133734, Abcam), anti‐SLC31A1 (ab129067, Abcam), anti‐Cleaved Parp antibody (ab32064, Abcam), anti‐Cleaved Caspase‐3 antibody (ab2302, Abcam), anti‐Bax antibody (ab32503, Abcam), anti‐Bcl‐2 antibody (ab32124, Abcam) and anti‐β‐actin antibody (ab8226, Abcam). The detailed steps of the assay have been described in our previous paper.³³ Briefly, the protein concentration was determined by BCA method, and then, total protein lysates were fractionated using SDS‐PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non‐fat milk and incubated with the above primary antibodies. Horseradish peroxidase‐conjugated secondary antibody was used to develop blots. The grey value was measured by ImageJ software.

Total cell extracts were prepared in ice-cold modified immunoprecipitate (IP) buffer (25 mM Tris-HCl [pH 7.5], 125 mM NaCl, 1% glycerol, 1 mM MgCl₂, 0.5% Triton, 0.5% Igepal CA-630, 0.5% sodium deoxycholate) supplemented with complete protease inhibitor cocktail (Roche), 5 mM NEM, 1 mM sodium orthovanadate (NaV), and 5 mM sodium fluoride (NaF) to inhibit phosphatases. For immunoprecipitation experiments, 2 mg of whole-cell lysates were incubated for 1–2 hr at 4°C with the appropriated amount of mouse monoclonal anti-frataxin (Abcam) or mouse monoclonal anti-RNF126 (Santa Cruz) antibodies per sample. The immunocomplexes were then absorbed for 1–2 hr at 4°C on prewashed protein glutathione Sepharose beads (GE Healthcare) and finally washed three times in wash buffer (25 mM Tris-HCl [pH 7.5], 125 mM NaCl, 1% glycerol, 1 mM MgCl₂, 0.5% Igepal CA-630) supplemented with complete protease inhibitor cocktail, 5 mM NEM, 1 mM NaV, and 5 mM NaF. After washing, immunocomplexes were resuspended in 2× Laemmli sample buffer, boiled for 5 min, resolved by SDS-PAGE, and analyzed by WB with rabbit polyclonal anti-frataxin (Abcam) or mouse monoclonal anti-RNF126 (Santa Cruz). Densitometric analyzes were performed using ImageLab software.