Ab180649

Cardiac muscle (25–50 mg) was homogenized as previously reported [22 (link)] and 5μg of homogenate loaded for SDS-PAGE. Proteins were separated on a 6%, 7.5%, 10% or 12% resolving gel as required to optimize for MW separation, and transferred to polyvinylidene difluoride membrane (Roche, Laval, QC, CA). The following commercially available antibodies were used: total OXPHOS antibody cocktail (Abcam, Cambridge, MA, USA, ab110413, 1:500,), eNOS (Abcam, ab5589, 1:1000), VEGF (Abcam, ab46154, 1:1000), HIF1α (Abcam, ab463, 1:1000), alpha tubulin (Abcam, ab40742, 1:5000), muscle RING finger protein-1 (MuRF1; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32920, 1:500), Muscle atrophy F-box (MAFbx; Santa Cruz, sc33782, 1:500), forkhead transcription factor-3a, Serine residue 253 (FOXO3a Ser253; Abcam, ab47285, 1:500), atrial natriuretic peptide (ANP; Abcam, ab180649, 1:500), BNP (Abcam, ab19645, 1:500) and beta-myosin heavy chain (β-MHC; Abcam, ab172967, 1:2000). All samples were detected from the same Western blot by cutting gels and transferring onto a single membrane to limit variability. Equal loading of protein was verified using Ponceau staining as well as constant alpha tubulin. All blots were quantified using enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON, CA) and quantified by densitometry (Alpha Innotech Fluorchem HD2, Fisher Scientific, Ottawa, ON, CA).

For Western blotting analysis, protein samples were extracted from the cells through protein extraction reagent (Thermo) and the concentration of protein samples was determined by BCA Quantitative Kit (Beyotime). Then, the difference of protein expression was analysed by Western blotting analysis. The antibodies used in this process were as follows: rabbit anti‐Myocardin (DF2434, Affinity), rabbit anti‐MBNL1 (ab45899, Abcam), mouse anti‐ACTN2 (ab9465, Abcam), rabbit anti‐ANP (ab180649, Abcam), rabbit anti‐TNF‐α (#3707, CST), rabbit anti‐p300 (#86377, CST) and rabbit anti‐GAPDH (#97166, CST). GAPDH expression was used as an internal control.