Antiphosphorylated akt ser473

Protein concentrations were determined with the BCA Protein Assay kit and a spectrophotometer (both by Thermo Fisher Scientific). Equal amounts of protein sample were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. The membranes were blocked with 5% skimmed milk in TBS buffer for 1 h. Proteins were then probed with anti-FLAG monoclonal antibody (Sigma, St. Louis, MO, USA), anti-DOK7 antibody (Abcam, Cambridge, MA, USA), monoclonal mouse anti-human GAPDH antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-Akt antibody (Sigma) and anti-phosphorylated-Akt (Ser473, Cell Signaling Technology, Danvers, MA, USA) and corresponding peroxidase-conjugated secondary antibody. Protein bands were visualised and analysed using Vilber Fusion Fx5 (Vilber, Marne La Vallée, France).

Protein of the TA muscle (or differentiated C2C12 cells) was extracted using lysis buffer containing 1% Triton X-100, 20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg ml⁻¹ leupeptin, 0.1 mM PMSF and a cocktail of protease inhibitors (Sigma, St Louis, MO, USA, Cat# P0044). 25 μg of protein per lane and prestained molecular weight markers (Bio-Rad, Hercules, CA, USA Cat# 161–0318) were separated with 12% SDS/PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking at room temperature for 1 h with a solution containing 3% bovine serum albumin (BSA, Fisher Scientific, Waltham, MA, USA, Cat# 137043) in 10 mM Tris-HCl (pH 7.5), 100 mM NaCl and 0.1% Tween-20 (TBST), membranes were incubated overnight at 4 °C with 1:500 rabbit polyclonal anti-phosphorylated-AKT (Ser473) (Cell Signaling, Danvers, MA, USA, Cat# 9271), rabbit anti-phosphorylated-Smad2/3 antibody (1:1000), or GAPDH (1:5000, Sigma, Cat# G9545), followed by 1:5000 horseradish peroxidase (HRP)-conjugated secondary antibody incubation. A chemiluminescent detection kit (PerkinElmer, Waltham, MA, USA, Cat# 203–100901) was used to develop the membranes.

Cells were lysed in cell lysis buffer (50 mM Tris‐HCl pH 7.5, 125 mM NaCl, 5 mM EDTA and 0.1% Triton X‐100) containing both 1% protease inhibitor and 1% phosphatase inhibitor cocktail (Sigma‐Aldrich, St Louis, MO, USA). Equal amounts of protein were separated on 10% SDS‐PAGE and then transferred to polyvinylidene fluoride membranes with an iBlot^®2 Gel Transfer Stack (Thermo Fisher Scientific). The membranes were incubated with primary antibodies at 4°C overnight. The primary rabbit antibodies used were anti‐ANXA10 antibody (1:500, NBP1‐90156SS, Novus Biologicals), anti‐phosphorylated Akt (Ser473) (/1:500, #4060, Cell Signaling Technology, Beverly, MA), anti‐phosphorylated Akt (Thr308) (1:500, #2965, Cell Signaling), anti‐Akt (1:1000, #9272, Cell Signaling), anti‐phosphorylated Erk1/2 antibody (Thr202/Tyr204) (1:500, #9101, Cell Signaling), anti‐Erk1/2 antibody (1:1000, #9102, Cell Signaling), anti‐β‐actin antibody (1:1000, #4970, Cell Signaling). The membranes were then probed with secondary antibodies for 90 min at room temperature. The secondary antibody was horseradish peroxidase‐conjugated donkey anti‐rabbit IgG (1:1000, NA934V, GE Healthcare, Little Chalfont, Buckinghamshire, UK).