Analysis of Smad 5 phosphorylation in PC3 cells. A and B; F-H. Protein and phosphorylation levels of Smad 5 were determined by Western blot analysis in nuclear (N), cytosolic (C) and total cellular (T) proteins isolated from PC3 cells (A and B) and PC3 cells knockdown of CD44 (F-H). 50 μg of indicated protein (A-D, F-H) was used for immunoblotting (IB) analyses. The blot in A was stripped and reprobed successively with p-Smad 5, GAPDH and histone antibodies (B-D). Similarly, the blot in F was stripped and reprobed twice simultaneously with GAPDH and histone antibodies (G and H). Immunoblotting with an antibody to GAPDH (C and G) and histone (D and H) was used as a control for normalization of cellular and nuclear protein, respectively. E. Confocal analysis of immunostained PC3 cells with Smad 5 (green) and p-Smad 5 (red) antibodies is shown. Distribution of both Smad 5 and p-Smad 5 is shown in the overlay panel. Scale bar-50 μm. The results shown are representative of three independent experiments.