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. 2013 Mar 5;7(3):647–668. doi: 10.1016/j.molonc.2013.02.008

Figure 4.

Figure 4

EVI1Del190–515 alters TGFβ‐mediated transcriptional regulation of the PAI‐1 promoter in ovarian cells. (A) Co‐immunoprecipitation of SMAD3 with EVI1 was performed using an anti‐FLAG antibody with lysates from cells expressing EVI1 and EVI1Del190–515 (as EGFP fusions) and FLAG‐SMAD3 followed by western analysis using EVI1 and SMAD3 polyclonal antibodies. The inputs represent 10% of the total. Based on densitometric analyses, between 1 and 5% of SMAD2/3 immunoprecipitated with EVI1. (B–C) Nucleofector transfection was performed on T29 cells with EVI1, EVI1Del427–515, or EVI1Del190–515 (5 μg) in combination with CAGA (1 μg) (B) or AP‐1 (1 μg) (C) using Renilla luciferase to normalize (0.05 μg). Cells were re‐seeded 6 h post‐transfection, allowed to adhere for 6 h, and serum starved/treated with 50 pM TGFβ. 50 pM TGFβ was chosen as being on the linear portion of the dose curve. The following day (24 h post‐transfection), cells were harvested in passive lysis buffer and assessed for luciferase activity using Dual Luciferase Assay kit.