Anti pten 9552

Cell lysates were prepared with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % Triton-X-100, 0.1 % SDS, 0.5% sodium deoxycholate) supplemented with 1x cOmplete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Pierce). Total protein concentration was measured using the Bio-Rad Protein Assay kit. Proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane. Protein-containing membranes were blocked with Odyssey Blocking Buffer (Licor) containing 0.1 % Tween 20. Western blots were performed with the following rabbit polyclonal antibodies, all purchased from Cell Signaling Technology (anti-RUNX2 #8486; anti-PTEN #9552, anti-AKT #4691; anti-pAKT-T308 #2965; anti-pAKT-S473 #4060). For purposes of protein normalization, mouse anti-β-actin antibodies were used (Abcam ab6267). All primary antibodies were used at a 1:1000 dilution. Secondary antibodies (IRDye® 800CW-labeled anti-rabbit; IRDye® 680RD-labeled anti-mouse) (Licor) were used at a 1:10000 dilution following the manufacturer’s recommendation. Resulting band intensity was calculated and quantified using the Licor Odyssey software.