FIGURE 6.

DPP-mediated nuclear localization of p-Smad1 in C3H10T1/2 is abrogated in the presence of KN-62. C3H10T1/2 cells were stimulated with rDPP (500 ng/ml) for 1 h. Cells were fixed and immunostained for p-Smad1. Immunofluorescence shows p-Smad1 predominantly located in the cytoplasm of untreated cells (A). With DPP stimulation, the predominant nuclear localization of p-Smad1 is observed. Arrows indicate nuclear localization of p-Smad1 (B). Diffused cytoplasmic staining of p-Smad1 was observed in cells pretreated with KN-62 alone (C). However, C3H10T1/2 cells stimulated with rDPP and KN-62 prevented nuclear localization of p-Smad1 (D). Bar, 10 μm. Nuclear and cytoplasmic proteins were extracted from C3H10T1/2 cells stimulated with DPP, and Western blotting was performed with anti-p-Smad1/5/8 and Smad1/5/8 antibody. The blots were stripped and reprobed with lamin A/C and tubulin (E). C, control.