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. 2010 Jun 14;30(16):4006–4021. doi: 10.1128/MCB.01600-09

FIG. 3.

FIG. 3.

BCL-3 is the only IκB protein that binds CtBP. (A) BCL-3 but not p52 binds CtBP in transfected cells. (Top) 293 cells were transfected with the indicated expression plasmids, and anti-FLAG immunoprecipitation (IP), followed by Western blotting (WB) with an anti-CtBP antibody, was carried out with the cell extracts. (Center and bottom) Western blot analyses with antibodies against FLAG and CtBP were also performed with the crude cell extracts. (B) Evidence for a ternary complex that includes p52, BCL-3, and CtBP. (Top panel) 293 cells were transfected with the indicated expression plasmids, and cell lysates were first subjected to anti-FLAG immunoprecipitation. A FLAG peptide was subsequently added in order to release the immunoprecipitates, which were then immunoprecipitated using the anti-BCL-3 antibody (lanes 1 to 6). The anti-FLAG immunoprecipitate from lane 7 was used directly for Western blotting with an anti-Myc antibody to detect Myc-CtBP, as were the anti-BCL-3 immunoprecipitates from lanes 1 to 6. (Center and bottom panels) Crude cell extracts were also subjected to Western blotting as indicated. (C and D) BCL-3, but not p52, p100, p105, or IκBα, binds CtBP. (Top) 293 cells were transfected with the indicated expression plasmids. Immunoprecipitation with an anti-Myc (C) or anti-FLAG (D) antibody was carried out with the cell extracts, followed by Western blotting with an anti-FLAG (C) or anti-HA (D) antibody (top panels). (Center and bottom) Western blot analyses with antibodies against FLAG (C and D), Myc (C), and HA (D) were also performed with the crude cell extracts. (E) Ectopically expressed CtBP binds LSD1 but not IκBα, IκBβ, IκBɛ, p100, or p105. 293 cells were transfected with FLAG-CtBP or an empty FLAG-expressing construct, as indicated (lanes 4 and 3, respectively), and anti-FLAG immunoprecipitation was carried out on the cell extracts. Western blotting using the indicated antibodies was subsequently performed on the immunoprecipitates (lanes 1 and 2) and on the cell extracts (lanes 3 and 4).