Serca2

Total cellular proteins were extracted using the RIPA lysis buffer, and a BCA assay kit (Beyotime) was used to quantify the protein concentrations. The same amounts of proteins (20 μg) were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% low-fat milk for 1 h, and then incubated with primary antibodies against SERCA1 (1:500; Affinity Biosciences), SERCA2 (1:5000; Abcam), SERCA3 (1:5000; Proteintech), IP3R (1:1000; Affinity Biosciences), PLCβ1 (1:1000; Proteintech), P2Y2 (1:1000; Affinity Biosciences), P2Y4 (1:1000; Affinity Biosciences) P2Y12 (1:2000; Abcam), Col1a1 (1:1000; Abcam), Runx2 (1:1000; Affinity Biosciences), Osx (1:1000; Bioss), β-catenin (1:1000; Proteintech), OPG (1:1000; Abcam), RANKL (1:1000; Proteintech), DKK1 (1:2000; Proteintech), NFATc1 (1:500; Santa Cruz), Calcr (1:1000; Proteintech), Ctsk (1:1000; Affinity Biosciences), TRAP (1:2000; Abcam), PPARα (1:1000; Affinity Biosciences), p-PPARα (1:1000; Affinity Biosciences) or β-actin (1:4000; Proteintech) overnight at 4  °C. The blots were incubated with HRP-conjugated goat-anti-rabbit secondary antibody (1:5000; Abcam) or HRP-conjugated goat-anti-mouse secondary antibody (1:5000; Abcam), and then visualized using an enhanced chemiluminescence system (Image Quant 350, GE Healthcare).

Tissue and cell lysates were prepared using RIPA buffer (20 mM Tris-Cl pH 7.5, 140 mM NaCl, 1 mM CaCl₂ and MgCl₂, 10 mM NaF, 1% NP-40, 10% glycerol, 2 mM Na-Vanadate, and 1 mM PMSF). Proteins were subjected to SDS-PAGE and were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Immunoblotting was performed with the following primary antibodies, according to the manufacturer’s protocol: KLF9 (Invitrogen; Cat: 701888; Clone name: 5H16L7; Dilution: 1:1000), KLF9 (Abcam; Cat: ab227920; Dilution: 1:1000), PGC1α (Millipore; Cat: AB3242; Dilution: 1:1000), UCP1 (Abcam; Cat: ab10983; Dilution: 1:1000), SERCA2(ABclonal; Cat: A11692; Clone name: ARC0679; Dilution: 1:1000), β-Actin (ABclonal; Cat: AC026; Clone name: ARC5115-01; Dilution: 1:20,000), ARG1 (ABclonal; Cat: A4923; Clone name: ARC1164; Dilution: 1:1000), SREBP1 (ABclonal; Cat: A15586; Dilution: 1:1000), HDAC1 (ABclonal; Cat: A0238; Dilution: 1:1000), HDAC2 (ABclonal; Cat: A2084; Dilution: 1:1000), SIN3A (Proteintech; Cat: 14638-1-AP; Dilution: 1:1000), STAT3 (Santa Cruz Biotechnology; Cat: sc-8019; Dilution: 1:50), P-STAT3 (Santa Cruz Biotechnology; Cat: sc-8059; Dilution: 1:50), p65 (Cell Signaling Technology; Cat: #8242; Clone name: D14E12; Dilution: 1:1000), P-p65 (Cell Signaling Technology; Cat: #3033; Clone name: 93H1; Dilution: 1:1000).

Muscle homogenates, purified isolated sarcolemmal vesicles and mitochondrial fractions were analyzed for total protein (BCA protein assay). Five micrograms of denatured protein from each sample were separated by electrophoresis on 7.5% and 12% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. Thereafter, membranes were blocked in 7.5% BSA and probed over night with commercially available antibodies against FAT/CD36 (rats; donated by Dr. Tandon; mice; Santa Cruz), FABPpm (donated by Dr. Calles-Escandon), COXIV (cytochrome c oxidase IV; Invitrogen), Cav-1 (caveolin-1; BD Biosciences), MCT1 (monocarboxylate transporter 1; Abcam), SERCA2 (sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; Thermo Scientific), AMPKα total and AMPKα Thr172 phosphorylation (Cell Signaling), ACC (acetyl-CoA carboxylase) and ACC Ser79 phosphorylation (Cell Signaling), extracellular signal-regulated kinase (ERK1/2) total and Thr202/Tyr204 phosphorylation (Cell Signaling). All blots were quantified using enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON) and quantified by densitometry (Alpha Innotech Fluorchem HD2, Fisher Scientific, Ottawa, ON).