P akt antibody

Western blot was performed following previously published method [29 (link)] to analyze the changes of specified proteins. Briefly, primary skeletal muscle fibroblasts were lyzed with RIPA buffer (Beyotime Bio, Shanghai, China) supplemented with proteinease inhibitor and phosphatase cocktail (Sigma, St. Louis, MO) and total proteins (40 μg) were resolved on 8% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked in 5% nonfat milk in TBST (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20) for 45 min, incubated with primary antibodies at 4 °C overnight, washed and incubated with proper horseradish peroxidase conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) at room temperature for 60 min before visualized with enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL). The primary antibodies used were Col I antibody, a-SMA antibody, CTGF antibody, TGF-β1 antibody, Fibronectin antibody, p-AKT antibody, AKT antibody, p-ERK antibody, PKC antibody, p-PKC antibody and β-Actin Antibody (sc-47,778) were purchased from Santa Cruz Biotech (Shanghai, China).

Detailed procedure for western blot was described elsewhere [21 (link)]. Primary antibodies used in this study included AFP, PTEN, E-cadherin, vimentin, VE-cadherin, AKT, Myc and GAPDH were obtained from Saier Co. (Tianjin China). p-AKT antibody was obtained from Santa Cruze and the ubiquitin antibody was obtained from Sigma. The secondary antibody goat anti-rabbit and goat anti-mouse were obtained from Sigma. GAPDH was used as the endogenous control to normalize the expression level of interest proteins.

Total proteins from each group were extracted by the use of NP-40 lysis solution (Beyotime, China). The concentration of the proteins was subsequently determined with the BCA Protein Assay Kit (Beyotime). After being electrophoretically separated by 15% SDS-PAGE, the total proteins transferred to PVDF membranes (Millipore, USA). Then, TBST buffer with 5% nonfat dry milk blocked them for 1 h. Primary antibodies p-FAK antibody (sc-11765, Santa Cruz, USA), FAK antibody (sc-557, Santa Cruz), p-mTOR antibody (sc-101738, Santa Cruz), mTOR antibody (sc-8319, Santa Cruz), p-AKT antibody (sc-135651, Santa Cruz), AKT antibody (sc-8312, Santa Cruz), p-ERK antibody (sc-7383, Santa Cruz), and ERK antibody (sc-135900, Santa Cruz) were added overnight at a dilution of 1 : 400 at 4°C. In comparison, a secondary antibody labeled with horseradish peroxidase (HRP) was used and incubated for 45 mins at 37°C. Color was developed with ECL luminescent solution (Millipore, USA), and after exposure, the results were analyzed by a gel imaging system. Gray scale analysis of protein bands was performed by ImageJ with β-actin as an internal protein.