FIG. 6.

Interactions with the CTD and colocalization with RNAP II. α, anti. (A) GFP-tagged T-U2AF65 and T-NLS proteins were immunoprecipitated from cell extracts and analyzed by Western blotting using the indicated antibodies (Ab). (B) Confocal microscopy of HeLa cells transfected with GFP-tagged T-U2AF65 and immunostained with H14 antibody. (C) In vitro pull-down assays for HA-tagged U2AF65 RS domain with GST-CTD or with GST alone as a control. Inputs show the purified Coomassie blue-stained recombinant proteins used in the pull-down assays. (D) HeLa cells were transfected with the HA-Rpb1 or HA-Rpb1ΔCTD constructs shown (top), and expression levels were assessed by Western blot analysis relative to antiactin antibody (bottom left). Cells were then cotransfected with T-U2AF65-GFP, and extracts were immunoprecipitated (IP) with anti-HA and probed for T-U2AF65-GFP by Western blotting with anti-GFP (bottom right). (E) The left panel shows doxycycline (DOX)-induced expression of HF-tagged Tat AD (T-NLS-HF) and T-RS-HF in stable HeLa T-Rex cell lines analyzed in whole-cell extracts with anti-FLAG. The right panel shows extracts bound and eluted from a GST-TFIIS column that were probed with the antibodies indicated. The asterisk indicates a cross-reacting protein.