Ac k antibody

For protein expression analysis, frozen tissues were homogenized in RIPA buffer containing protease inhibitors. Protein-matched samples (Bradford assay) were electrophoresed (SDS-PAGE) and then transferred to nitrocellulose (NC) membranes. The NC membranes were blocked with 5% skim milk in TBS (25 mmol/L Tris base and 150 mmol/L NaCl) for 2 h at room temperature and then incubated with the following primary antibodies (1:1,000 diluted) at 4°C overnight. SREBP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ac-K antibody (Cell Signaling Technology, Beverly, MA, USA), ACC antibody (Thermo Fisher, Waltham, MA, USA), FAS antibody (Thermo Fisher), GAPDH antibody (Thermo Fisher), and SCD1 antibody (Abcam, Cambridge, UK). The membranes were incubated with secondary antibodies (1:5,000 diluted) at room temperature for 1 h and then washed three times for 10 min each in TBST. The target proteins were detected with ECL plus detection reagents (Amersham, Pittsburgh, PA, USA). The expression levels were quantified using optical densitometry and the ImageJ software (http://rsbweb.nih.gov).

Protein extracts were prepared in RIPA lysis buffer (Thermo Fisher Scientific #89900) from snap-frozen heart tissues. Protein concentration was measured by BCA assay (Thermo Fisher Scientific #23227). Immunoprecipitation of tissue/cell lysates was performed in RIPA buffer, with equal amounts of protein used for immunoprecipitation assay with rabbit acetyl-lysine (Ac-K) antibody (Cell Signaling Technology #9441) and protein G agarose beads (Cell Signaling Technology #37478) overnight at 4°C. Beads were washed 4 times with RIPA buffer, and then eluted directly with LDS sample buffer (Thermo Fisher Scientific #B0007) at 95°C for 5 mins. Protein electrophoresis was performed on 4–12% Bis-Tris NuPage gels (Thermo Fisher Scientific #NW04120BOX). The Bio-Rad Trans-Blot Turbo Transfer System was used for protein transfer to nitrocellulose membranes. The related secondary antibodies used were from LI-COR Biosciences, and blots were quantified using Image J software (NIH). GCN5L1 antibody was used as previously reported.¹⁰ (link) Oxphos Cocktail antibody (#ab110413) was from Abcam. The TFAM (#8076S), α-tubulin (#2148), GAPDH (#5174), and VDAC (#4866) antibodies were from Cell Signaling Technologies.

For protein expression analysis, frozen tissues were homogenized in RIPA buffer containing protease inhibitors. Protein-matched samples (Bradford assay) were electrophoresed (SDS- PAGE) and then transferred to nitrocellulose (NC) membranes. The NC membranes were blocked with 5% skim milk in TBS (25 mmol/L Tris base and 150 mmol/L NaCl) for 2 h at room temperature and then incubated with the following primary antibodies (1:1000 diluted) at 4°C overnight. SREBP1 antibody (Santa Cruz Biotechnology, CA, USA), ac-K antibody (Cell Signaling Technology, MA, USA), ACC antibody (Thermo Fisher, MA, USA), FAS antibody (Thermo Fisher), GAPDH antibody (Thermo Fisher), and SCD1 antibody (Abcam, Cambridge, UK). The membranes were incubated with secondary antibodies (1:5,000 diluted) at room temperature for 1 hour and then washed three times for 10 min each in TBST. The target proteins were detected with ECL plus detection reagents (Amersham, Pittsburgh, PA, USA). The expression levels were quantified using optical densitometry and the ImageJ software (ImageJ software; http://rsbweb.nih.gov).