Figure 2

BHEXIM1 interacts directly with B-cyclin T1. (A) Co-IP of BHEXIM1 and B-cyclin T1. 293 T cells were co-transfected with Flag-B-cyclin T1 and either an empty vector (lane 1) or HA-BHEXIM1 (lane 2). BHEXIM1 was immunoprecipitated with rabbit anti-HA agarose beads, and the immunoprecipitates were detected by western blot using mouse anti-Flag (top) or mouse anti-HA (middle) antibodies. The amount of B-cyclin T1 in each cell lysate was checked using a mouse anti-Flag antibody (bottom). (B) Reciprocal Co-IP of BHEXIM1 and B-cyclin T1. 293 T cells were co-transfected with HA-BHEXIM1 and either an empty vector (lane 1) or Flag-B-cyclin T1 (lane 2). B-cyclin T1 was immunoprecipitated with mouse anti-Flag agarose beads, and the immunoprecipitates were detected by western blot using rabbit anti-HA (top) or mouse anti-Flag (middle) antibodies. The amount of BHEXIM1 in the cell lysates was checked using a rabbit anti-HA antibody (bottom). (C) In vitro interaction between BHEXIM1 and B-cyclin T1. The purified BHEXIM1 protein was incubated with GST or GST-B-cyclin T1 agarose beads. GST pulldown was then performed, and precipitates were detected by western blot using an anti-His antibody. Purified His-BHEXIM1, GST and GST-B-cyclin T1 used in the pulldown assay were detected by Coomassie blue staining.