Protein Extraction and Western Blot Analysis

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).