The effect of PKC and integrin αv inhibitor on the phosphorylation of Smad 5 and RUNX2 localization in the nuclei. A. Analysis of interaction of p-Smad 5 with RUNX2. Equal amount of total cellular and nuclear proteins were immunoprecipitated with a RUNX2 antibody and immunoblotted with a p-Smad 5 antibody (A, top panel). Subsequently, the blot was reprobed sequentially with a RUNX2 (middle panel) and p-Serine (bottom panel) antibody after stripping. B. Effect of SiRNA to Smad 5 on the nuclear levels of RUNX2. Time-dependent effect of SiRNA (Si) nucleotides on Smad 5 levels at 48 and 72 h is shown. Equal amount of nuclear proteins were immunoblotted sequentially with antibodies to Smad 5, RUNX2 and nucleoporin after stripping. Scrambled RNAi nucleotide (Sc) transfected cells were used as controls (lane 1). C. Effects of PKC and integrin αv inhibitors (lanes 2 and 3) on the phosphorylation of Smad 5. Untreated (-) PC3 cells were used as control. Total cellular (T) lysate proteins were immunoblotted with a p-Smad 5 antibody. D. Effects of PKC and integrin αv inhibitors (lanes 2 and 3) on the nuclear localization of RUNX2. Untreated (-) PC3 cells were used as control. Nuclear lysate proteins (N) were immunoblotted with a RUNX2 antibody. B-D: Loading control antibodies to GAPDH (C) and nucleoporin (B and D) were used to estimate relative amounts of total and nuclear proteins loaded in each lane. The results shown are representative of three independent experiments.