FIG. 3.
Bim phosphorylation. (A) Immunoblot analysis using the Bim antibody with lysates from parental Baf-3 cells (pt) (lanes 1 and 2) or cells overexpressing BimEL and BimL (lanes 3 and 4) cultured in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of IL-3. To demonstrate an extra band comigrating with BimL, we added five times more cell extract to lane 1 than to the other lanes. (B) Lysates from Baf-3 cells overexpressing BimEL and BimL proteins were immunoprecipitated (IP) with preimmune serum (lane 1) or Bim antibody (lanes 2 to 4). After treatment with CIAP in the absence (lane 3) or presence (lane 4) of phosphatase inhibitor, precipitants were separated with SDS-PAGE and Bim proteins were then detected with Bim antibody. (C) Baf-3 cells overexpressing BimEL protein were cultured in medium containing [32P]orthophosphate in the presence (lanes 1 and 2) or absence (lane 3) of IL-3 for 4 h. Immunoprecipitates obtained by preimmune serum (lane 1) or Bim antibody (lanes 2 and 3) were separated by SDS-PAGE. (D) Lysates from Baf-3 cells overexpressing BimEL protein were immunoprecipitated with preimmune serum (lane 2) or antibody against Bim (lane 3), Bcl-2 (lane 4), or Bcl-x (lane 5). After immunoprecipitation and SDS-PAGE followed by blotting, Bim proteins were detected with Bim antibody. Lane 1 is a positive control to detect BimEL protein in whole-cell lysates.