FIG. 7.
Subcellular distribution of p21Cip1 in dependency on Thr 145 phosphorylation. (A) Immunostaining of human endothelial cells transfected with p21 wt or the Thr 145 constructs. Immunocytochemistry was performed using anti-myc antibody and counterstained with DAPI (n = 4). Due to the limited transfection efficiency, not all cells reveal FITC staining. (B) Immunoblot analysis of p21Cip1 protein levels in the nuclear versus cytoplasmic fraction of HUVEC overexpressing p21 wt and Thr 145 constructs. (C) Densitometric analysis of four individual experiments as in panel B. Data are mean ± SEM; *, P < 0.05 versus the wild type and T145A. (D) Relative subcellular distribution of Akt in nucleus and cytosol of HUVEC transfected with p21Cip1 constructs or vector. A representative result of three individual experiments is shown. In the results shown in panels B and D, immunoblots were reprobed with anti-topoisomerase antibody to verify separation of the nuclear fraction and as a loading control. WB, Western blotting.