Figure 3.

TFII-I is phosphorylated upon treatment of cells with TGFβ1. (A) Phosphoprotein spots in which TFII-I was identified are shown in selected areas of images of gels with ³²P-labeled proteins, which were prepared from MCF-7-vector, MCF-7-Flag-Smad2, and MCF7-myc-Smad3 cells. Migration positions of TFII-I-containing spots P10.1, P10.2, and P10.3 are indicated and are shown by arrows in two panels. Due to the presentation of gel images at similar intensities, borders for some of the software-designated spots may not be clearly recognized in panels. pH gradient is shown on the bottom of one of the images. Type of cells and time of treatment with TGFβ1 are shown on the side and on the top of the images, respectively. Quantification of total radioactivity in all three TFII-I-containing spots (summarized total activity in P10.1, P10.2, and P10.3 spots for one experimental condition) in cells treated with TGFβ1, or not, as indicted, is shown below the images. Note the difference in total radioactivity at 0-h time point. TGFβ1 treatment and cell lines are indicated. (B) Total phosphorylation of TFII-I was analyzed by immunoprecipitation of ³²P-labeled myc-TFII-I with anti-myc antibodies. Phosphorylation of transfected myc-TFII-I in MCF-7-vector cells upon treatment of cells with TGFβ1 (5 ng/ml) for 1, 4, and 24 h is shown (top). Expression of myc-TFII-I was monitored by immunoblotting of the same membrane with anti-myc antibodies (B; bottom). (C) Phosphorylation of endogenous TFII-I in MCF-7-vector, MCF-7-Flag-Smad2, and MCF-7-myc-Smad3 cells was evaluated by immunoprecipitation of TFII-I with antibodies specific to endogenous protein from cells metabolically labeled with [³²P]orthophosphate. Metabolic labeling of cells with [³²P]orthophosphate was performed, as described in the Materials and Methods. Expression of myc-TFII-I was monitored by immunoblotting of the same membrane with anti-myc antibodies. Migration position of ³²P-labeled and immunodetected myc-TFII-I are shown by arrows. Representative experiments out of three (B and C) performed are shown.