FIG. 6.

CoREST and LSD1 bind BCL-3 through its N-terminal domain. CtBP and LSD1 play roles in the repressing abilities of BCL-3. (A and B) The PVDLR motif of BCL-3 is required for binding to LSD1 but not to CoREST. (Top) Anti-BCL-3 (A) or anti-FLAG (B) immunoprecipitates (IP) with cell extracts derived from 293 cells transfected with the indicated expression plasmids were subjected to Western blot analysis (WB) with an anti-FLAG (A) or anti-LSD1 (B) antibody. (Center and bottom) Crude cell extracts were subjected to Western blotting with antibodies against BCL-3, LSD1, and FLAG, as indicated. (C) The association of BCL-3 with LSD1 is impaired in CtBP-depleted cells. (Top two panels) 293 cells were first transfected with the indicated siRNA targeting either GFP (negative control) or CtBP, as indicated. The resulting cells were transfected with the indicated expression vectors, and cell extracts were subjected to immunoprecipitation with an anti-FLAG antibody, followed by Western blot analysis with an anti-LSD1 or anti-p50 antibody. (Bottom three panels) Crude cell extracts were subjected to Western blotting with an antibody against LSD1, p50, CtBP, or FLAG, as indicated. (D) CtBP and LSD1 contribute to the repressing abilities of BCL-3. (Top) Control or BCL-3-expressing HaCat cells were transfected with a siRNA targeting GFP (control) (lanes 1 and 2) or with a siRNA targeting CtBP (left) or LSD1 (right) (lanes 3 and 4), and cell extracts were subjected to Western blotting with an antibody against Hsp90, BCL-3, CtBP, or LSD1. (Bottom) Total mRNA levels of PLAUR and RAB7L1 were quantified by real-time PCR under all experimental conditions (control or BCL-3-expressing cells, as indicated). The abundance of each transcript in cells infected with the control lentivirus was set to 1, and their levels under the other experimental conditions were relative to that after normalization with 18S rRNA. Data from three independent experiments (means ± standard deviations) are shown.