FIG. 5.

PKC δ directly phosphorylates Fli1 at threonine 312. (A) Cultured dermal fibroblasts and human microvascular endothelial cells were stained with anti-Fli1 antibody. Positive signals were developed with diaminobenzidine. (B) 293T cells were transfected with TβRI T204D or empty vector for 48 h and then cytoplasmic extracts (500 μl of buffer A) and nuclear extracts (500 μl of buffer C) were prepared. In order to evaluate the ratio of PKC δ localized in the cytoplasm (C) and nucleus (N) accurately, a 10% volume of each extract was subjected to immunoblotting with anti-PKC δ antibody. To confirm that the cytoplasm and nucleus were properly separated, the levels of β-actin and lamin A/C were determined. The bottom panel shows the relative expression levels of PKC δ in each compartment. (C) 293T cells were transfected with the indicated expression vectors for 48 h. Nuclear extracts and whole-cell lysates were prepared under the same conditions. Tagged-Fli1 was precipitated from nuclear extracts with streptavidin-coupled agarose beads (SA beads), and the precipitates were subjected to immunoblotting using anti-PKC δ antibody and anti-calmodulin binding peptide antibody. The levels of PKC δ in the nuclear extracts and the levels of TβRI T204D in the whole-cell lysates were determined by immunoblotting. WT, wild type. (D) Nuclear extracts (NE) and whole-cell lysates were prepared under the same conditions. Nuclear extracts were subjected to DNA affinity precipitation (DNAP) with COL1A2 EBS oligonucleotide. The levels of PKC δ in the precipitates were determined by immunoblotting. (E) Streptavidin bead-bound tagged Fli1 prepared from 293T cells was incubated with recombinant PKC δ-GST fusion protein. In order to activate PKC δ, phosphatidyl serine and phorbol ester were added to the reaction. The whole reaction was subjected to immunoblotting using anti-phospho-Fli1 (Thr312) antibody. The levels of tagged Fli1 and PKC δ were confirmed by immunoblotting on the same membrane.