Fig. 2. Binding of Six proteins to Grg proteins in vitro and in vivo. (A) Six family proteins bind to Grg5 in vitro. Coomassie staining of a gel containing GST (0.2 µg) and GST-Grg5 fusion proteins (0.2 µg) used in the GST pull-down experiments is shown on the top row. The input lane shows 10% of the total protein used in the GST pull-down experiments. Full-length Six3 migrated as a 37 kDa protein. The faster migrating bands may have been shorter forms of Six3 that originated from internal translation start sites in the Six3 transcript. (B) Six family proteins interact in vitro with mouse Grg4 and with Drosophila Groucho. Input lane shows 10% of the total [³⁵S]methionine-labeled proteins used in the GST pull-down experiments. Specifically, mouse Six3 and Six6 protein bound to GST-Grg4 fusion protein (GST-Grg4 lane) but not to GST alone (GST lane). Drosophila Optix bound only weakly to GST-Grg4 fusion protein, whereas Six3_F88E mutant protein did not. Fly Groucho strongly bound to mouse Six3 (lower panel). (C) Mouse Six3 bound to Grg4 in mammalian cells. NIH3T3 cells were transfected with either Six3 expression vector alone (lane 1) or Six3 expression vector and Flag-tagged Grg4 (lane 2). Co-immunoprecipitation (IP) was performed with anti-Flag antibody, and precipitated Six3 was detected with rabbit anti-mouse Six3 antibody (lane 2). Input lane shows 10% of the total protein used in the IP experiment. Six3 and Flag-Grg4 proteins in the crude cell lysate underwent western blot analysis. (D) Fly Groucho immunoprecipitated with mouse Six3. NIH3T3 cells were transfected with either Flag-tagged Groucho expression construct alone (lane 1) or Flag-tagged Groucho expression construct and Six3 expression vector (lane 2). Immunoprecipitation was carried out with anti-Six3 antibody. The precipitated Flag-tagged Groucho protein underwent western blot analysis with anti-Flag antibody. The input lane shows 10% of the total cell lysate used in the IP experiment. Flag-Groucho and Six3 in the crude cell lysate was subjected to western blot analysis.

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