SET overexpression increases Gli1-mediated transcription. (A) The effect of SET on Gli1-mediated transcription. To determine Gli1-mediated transcription upon SET overexpression, 12xGliBS-luc and Gli1 expression plasmids were cotransfected with a human Flag-SET expressing construct in HEK 293 cells. (B) The effect of SET on Sufu inhibition of Gli1-mediated transcription. HEK 293 cells were transfected with 12xGliBS-luc reporter with the indicated amounts of plasmids encoding Gli1 (100 ng), Flag-SET (100 and 200 ng) and Sufu (10 ng). pRL-TK expresses Renilla luciferase and was included in all samples to normalize the transfection efficiency. Experiments were repeated three times and samples were analyzed in triplicates, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Expression of GST-SET and GST-SAP18 recombinant proteins. Bacterially expressed glutathione-S-transferase-tagged SAP18 (GST-SAP18), GST protein and baculovirus expressed GST-SET (approximately 2–3 μg each) were immobilized on glutathione-Sepharose beads and analyzed by 12% SDS-PAGE. An original uncropped image of panel C is shown in Supplementary Fig. 2S. (D) GST-SET pull down experiments. GST-SET or GST was immobilized on glutathione-Sepharose beads and incubated with cell extracts expressing GFP-SAP18 or GFP. (E) GST-SAP18 pull down experiments. GST-SAP18 or GST was immobilized on glutathione-Sepharose beads and incubated with cell extracts expressing GFP-SET or GFP. The beads and the supernatant of the pull down assays were analyzed by western blot using the anti-GFP antibody. Original uncropped Western Blot images of panels D and E are shown in Supplementary Fig. 2S.
|
