Fig. 3

T-Antigen inhibits SRSF1 transcription through association with the promoter region. a. Effects of T-antigen and agnoprotein on SRSF1 transcription were tested by co-transfection studies. T98G cells were transiently transfected with pLuc.SRSF1(−1000) reporter plasmid and increasing concentrations of T-antigen, agnoprotein, and GFP expression plasmids. After 48 h, cell extracts were collected and analyzed for luminescence and normalized to protein concentrations. Relative luciferase activities were shown as bar graph. Standard deviations were calculated from three independent experiments. A representative panel of images of GFP expression from live cells transfected with increasing concentrations of pLEGFPC1 plasmid was also shown in the upper panel of the bar graph. b. Western blot analysis of whole cell extracts prepared in parallel to the samples in panel a. c. T-antigen is associated with SRSF1 promoter region. T98G cells were either transfected with T-antigen or left untransfected and proteins were cross-linked to DNA using formaldehyde, followed by ChIP analysis using antibody against T-antigen. Following immunoprecipitation, beads were eluted and underwent subsequent DNA purification. Purified DNA was analyzed by PCR for the −1000 to + 49 promoter region of SRSF1. In lane 2, the plasmid pLuc.SRSF1(−1000) was used as template and loaded as positive control. In lanes 5 and 6, samples from cells with and without T-antigen were subjected to immunoprecipitation by anti-T-Antigen antibody (IP-T-ag). In lanes 3 and 4, samples from cells with or without T-antigen expression were also subjected to immunoprecipitation by normal mouse serum (IP-NMS) as controls