Sc 33782

Placenta samples were homogenised in protein extraction buffer (100 mM Tris Base, 10 mM Na₄P₂O₇, 100 mM FNa, 1 mM Na₃ VO₄, 10 mM EDTA, 2 mM PMSF, 0.1 mg/mL aprotinin, 1 % Triton X-100, pH 7.4) followed by centrifugation at 10,000 × g for 15 min at 4 °C. Placental proteins (40 μg) were resolved by 12 % SDS-PAGE at 90 V for 1 h, followed by transfer onto 0.45-μm nitrocellulose membranes at 300 mA for 2 h. The membranes were blocked with 5 % non-fat dry milk in Tris-buffered saline (pH 7.5) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies against the 20S, 19S and 11S proteasome subunits (#PW8165, #PW8195, Affinity, USA; 1:1500 dilution) as well as MuRF-1 (#SC-32920) and MAFbx (#SC-33782; both from Santa Cruz Biotechnology, Heidelberg, Germany; 1:200 dilution). Immunoreactivity was detected by sequentially incubating the membranes with specific secondary antibodies (1:10,000 dilution, Cell Signaling Technology) for 1 h at room temperature and visualised using a chemiluminescence detection system. The blots were scanned using a gel image capture system to quantify differences via densitometry (Alliance 2.7 system, Alliance 1D capture software, and UVIBand 12.14 analysis software; UVITEC, Cambridge, UK). The levels of all detected proteins were reported relative to the level of 34-kDa GAPDH.

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).