Akt pser473

Samples of 10 ~ 50 μg total protein were subjected to western blot analysis using polyclonal antibodies to phosphorylated Akt (Akt-pSer⁴⁷³), Akt (Cell Signaling, Beverly, MA), HO-1 (Enzo Life Sciences, Farmingdale, NY), COX IV (Abcam, Cambridge, MA), α-Tubulin (Abcam), and β-actin (Sigma-Aldrich). Protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (PerkinElmer, Waltham, MA). Band intensities were quantified by densitometry using Image J program.

Cell pellets were lysed in lysis buffer (20mM Tris (pH8), 150mM NaCl, 0.5% NP-40, 1mM EDTA) supplemented with protease inhibitors (cOmplete EDTA-free protease inhibitor cocktail; Roche) and phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044; Sigma). Lysates were heated at 70°C for 10min in NuPAGE LDS sample buffer. Proteins were separated by SDS-PAGE on a Bolt 12% Bis-Tris Plus or a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membrane in NuPAGE transfer buffer plus 7.5% methanol. The membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 (TBST) plus 5% milk powder then incubated with primary antibodies: pan-14-3–3 antibody (8312S, Cell Signaling Technology), V5 antibody (R96025, Invitrogen), 14-3-3γ (Cell Signaling Technology, 5522), FOXO3a (Cell Signaling Technology 12,829), FLAG (Sigma, F1804), HSP90 (Cell Signaling Technology, 4877S), pan-AKT (Cell Signaling Technology, 2920S), AKT-pSer473 (Cell Signaling Technology, 4060S), FOXO3a-pSer253 (Cell Signaling Technology, 9466S), beta-actin (Sigma Aldrich, A5441), YAP (Cell Signaling Technology, 12395S), p53 (Santa Cruz, sc-126). Membranes were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies and developed using Amersham ECL Western Blotting Detection Reagents.

Western blot analysis was performed on flash-frozen LV tissue samples as previously described (26 (link), 32 (link)). Briefly, we extracted protein from LV tissues and performed immunoblotting for various proteins using the following primary antibodies: SERCA2a, NCX1 (Thermo Fisher Scientific), PLN-P^Ser16, total PLN (Badrilla Ltd), Akt-P^Ser473, Akt-P^Thr308, total Akt (Cell Signaling), AMPK-P^Thr172 and total AMPK (Cell Signaling) and subsequently incubated with HRP conjugated secondary antibodies respectively.