Effects of CD44 knockdown on RUNX2 expression (mRNA and protein) and distribution in PC3 cells. A. The expression levels of RUNX2 mRNA was determined by real-time PCR analysis and normalized relative to GAPDH expression. Bar represents the mean ± SEM of three different experiments. *p <0.01 vs. untransfected (−) and transfected PC3 cells with scrambled ShRNA construct (Sc) and vector DNA (V). B and C. Equal amount of lysates (20 μg protein) made from PC3 cells untransfected (−) and transfected with scramble (Sc) and ShRNA CD44 constructs (492 and 801) were used for immunoblotting analysis with an antibody to RUNX2. Immunoblotting with an antibody to GAPDH (C) was used as a loading control. D and E. PC3 cells were analyzed for the phosphorylation of RUNX2 in total cellular (T) and nuclear (N) lysates by immunoblotting of RUNX2 immunoprecipitates with antibodies to RUNX2 (D) and phospho-serine (E; p-Serine). The results shown are representative of three independent experiments.