Gapdh and β actin

Snap-frozen LV tissue samples were homogenized and lysed in a buffer containing: 25 mM Tris–HCl, 150 mM NaCl, 2 mM EGTA, 5 mM EDTA, 0.5% NP-40 with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, UK). For cell lysates, the buffer composition was: 25 mM Tris–HCl, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate with protease and phosphatase inhibitor cocktails. Protein concentration was estimated using Bradford reagent (Sigma-Aldrich, UK). Tissue homogenates were separated by SDS/PAGE and transferred onto nitrocellulose membranes. Membrane fractions were obtained by centrifugation of heart lysates¹³ (link) and further processed as described earlier. The following primary antibodies were used: phospho-Akt (S473), total Akt, phospho-Erk1/2 (Thr202/Tyr204), total Erk1/2, phospho-ribosomal protein S6 (S235/236), total ribosomal protein S6, phospho-Src (Tyr416), total Src, total PP2Ac (all Cell Signaling); Nox2 (BD Transduction); Nox4¹⁴ (link); eIF4E-BP1, caveolin-3 and phospho-PP2Ac (Tyr307) (Abcam); p47phox (EMD Millipore/Upstate); GAPDH and β-actin (Sigma). Imaging and densitometric quantification were undertaken either using enhanced chemiluminescence or with an Odyssey Li-Cor imaging system (Li-Cor Biosciences, UK).

N/TERT-1 grown in 2D or differentiated in 3D organotypic cultures (total cultures) were lysed with Lysis Buffer containing 50 mM Tris, HCl pH 8.0, 150 mM NaCl, 1% Triton X-100 and protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was measured according the Bradford method (BioRad, Munich, Germany). The protein solution was diluted in NuPage loading buffer (Invitrogen, Carlsbad, CA, USA) and 30 or 60 µg per line loaded on 4–12% Bis-Tris NuPage pre-casted gels (Invitrogen, Carlsbad, CA, USA). The separated proteins were transferred on nitrocellulose membrane using iBlot semi-dry transfer apparatus. Membranes were blocked with blocking buffer (5% skimmed milk, 0.1% Tween 20 in PBS) for 1 h at room temperature and incubated with primary antibody against p21 (Upstate, Temecula, CA), COX-2 (Millipore, Temecula, CA) 1∶500; Phospho-p38Tyr^180/182 (Cell Signaling, Denver, MA, USA) 1∶1000; GAPDH and β-actin 1∶5000 (Sigma-Aldrich, St. Louise, MO, USA) overnight at 4°C. The membranes were washed with 0.1% PBS-T and incubated with secondary HRP- conjugated antibody (ECL™ Anti-Mouse/Anti Rabbit IgG, GE Healthcare, Little Chalfont, UK) for 1 h at room temperature. After washing with 0.1% PBS-T membranes were incubated with SuperSignal ECL (Thermo Scientific, Rockford, IL, USA) and developed on X-ray sensitive film.

The cell or gastrocnemius muscle tissue was homogenized in a lysis buffer containing cOmplete™ protease inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN, USA) and protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA; Oakville, ON, Canada) and subsequently centrifuged. The BCA Protein Assay Kit (Bio-Rad, Hercules, CA, USA) was employed to determine the protein concentrations, ensuring an equal protein concentration across all experimental groups. The equivalent protein concentrations from each group were then subjected to SDS-PAGE. Following electrophoresis, the membranes were blocked with 5% skim milk and incubated with primary antibodies overnight. The primary antibodies utilized for Western blot analysis included IGF, p-AKT, t-AKT, p-mTOR, mTOR, p-PI3K, t-PI3K, MyoD, Myogenin, MuRF1, p-FOXO3a, FOXO3a, Sirt1, and PGC1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as GAPDH and β-actin (Sigma-Aldrich, CA, USA) for use as a loading control. Subsequently, the membranes were incubated with the corresponding secondary antibodies to visualize the protein bands using an LAS3000 luminescent image analyzer (Fuji Film, Tokyo, Japan). β-actin served as the loading control, and Image J software (National Institute of Health, Bethesda, MD, USA) was employed for quantitative analysis.