Noxa expression inhibits Mcl-1 binding to Bim displaced from Bcl-xL and Bcl-2. 8226/S, MM.1s, and KMS11 cell lines were treated with indicated concentrations of ABT-737 for 24 hours and lysates generated as described in “Western blot analysis.” (A) Mcl-1 and (B) Bcl-xL and Bcl-2 were immunoprecipitated using specific monoclonal antibodies. Immunoprecipitates were sequentially probed with pAbs for Bim and Noxa as well as for Bcl-2, Bcl-xL, and Mcl-1. Immunoprecipitates contain 60% of input. (C) The total amount of Bim was assayed by Western blot in lysates from control and treated samples of 8226/S, KMS-11, MM.1S. (D) Twenty-four hours after transfection with si(-) or siNoxa, expression of Noxa and Mcl-1 was determined by Western blot (right panel). The remaining lysate was subjected to immunoprecipitation with Mcl-1, Bcl-xL, or Bcl-2 followed by Western blot analysis of Bim, Mcl-1, Bcl-xL, and Bcl-2 (left panel). (E) The bands from 2 independent experiments were quantified using ImageJ software Version 1.45r, and the graph represents the average ratio of relative intensity of indicated bands.
