FIG 3.

The phosphomimetic S2D mutation in BCL11B inhibits NuRD recruitment. (A) Schematic drawing of the full-length BCL11B proteins tested. (B) The interaction with MTA1 is impaired by the S2D phosphomimetic mutation of BCL11B. HEK293T cells were transfected with the indicated combination of expression vectors, and Co-IP assays followed by immunoblotting with the indicated antibodies were performed. (C) The BCL11B-MTA3 interaction is also strongly reduced by the S2D phosphomimetic mutation. A similar Co-IP experiment was performed with HEK293T cells but with the FLAG-MTA3 expression vector. Relevant pieces of the membranes were cut (around the 80-kDa marker) to separate the MTA3 and BCL11B proteins and probed with anti-FLAG antibodies. (D) Interaction of wild-type (WT), S2A, S2D, and ΔMSRKKQ BCL11B with endogenous MTA1 proteins. Total extracts of HEK293T cells transfected with the indicated plasmids were analyzed by Co-IP with anti-FLAG antibodies and immunoblotted with MTA1 and FLAG antibodies. (E and F) Interaction of the S2A and S2D BCL11B point mutants with endogenous NuRD components. Co-IP experiments followed by immunoblotting with the indicated antibodies were performed as in panel D with the S2A and S2D (E) or S2D (F) BCL11B mutants.