FIGURE 5.

Increased binding of BIM to MCL-1 causes sunitinib and S63845 synergism. (A) Western blot analysis of the unbound fraction after MCL-1 immunoprecipitation. (B) Immunodetection of MCL-1 and BIM initial expression in cell lysates after 16 h of incubation with sunitinib 100 nM, and 2 h incubation with S63845 100 nM in the specified conditions. Quantification of optical density for each protein was normalized to actin and fold-change was calculated comparing to protein expression in the control condition. (C) Western blot of the immunoprecipitated fraction was used to study the interaction between MCL-1 and BIM after treatment with sunitinib 100 nM and 2 h with S63845 100 nM. To quantify this binding, BIM optical density was normalized to MCL-1 optical density and fold-change was calculated comparing to protein expression in the control condition. All results are expressed as the mean ± SEM of at least three biologically independent replicates. Statistical significance was calculated using Student’s t-test compared to control condition and considering *p < 0.05 and **p < 0.01.