BIM phosphorylation and binding to MCL1. (A) CLL samples were treated with 20 μg/mL anti-IgM for the indicated times, and expression of MCL1 and β-actin was analyzed by immunoblotting. Figure shows results obtained with 3 CLL samples and is representative of a total of 6 samples. (B) Ramos cells were incubated in the presence or absence of anti-IgM for 30 minutes. Lysates were prepared and immunoprecipitations (IP) performed using an anti-BIM antibody or an isotype control (IC). The immunoprecipitates and a portion of the lysates were analyzed by immunoblotting using antibodies specific for MCL1 or BIM, as indicated. Open arrowheads indicate phosphorylated BIM isoforms. (C) Quantitation of BIM_EL phosphorylation and the proportion of MCL1 associated with BIM in Ramos cells treated with anti-IgM for 30 minutes. The amount of MCL1 bound to BIM in unstimulated cells was set to 1.0. Data are derived from 6 independent experiments (mean ± SD). (D) Ramos cells were incubated for 30 minutes with or without IgM stimulation. The IP was performed using rabbit anti-MCL or the appropriate IC, and the precipitates/cell lysates were probed with anti-BIM and anti-MCL1 antibodies. (E) IP was performed with a mouse anti-MCL1 antibody or the appropriate IC, to enable immunoblotting with a rabbit S⁶⁹ phospho-BIM–specific antibody. (D-E) Data are representative of 3 independent experiments.