Figure 7.

EGF modulates the interaction between KSR1 and calmodulin.A, KSR1+/+ MEFs were serum-starved for 16 h, followed by incubation with 100 ng/ml EGF for 0, 1, 5, or 10 min. Cells were lysed in buffer containing 1 mM Ca²⁺ and 1 mg protein lysate was immunoprecipitated with anti-KSR1 antibody. A sample precipitated with mouse IgG was processed in parallel as negative control. Both unprocessed lysate (Input) and immunoprecipitates (IP) were resolved by western blotting and blots were probed with anti-calmodulin (CaM) and anti-KSR1 antibodies. B, equal aliquots of protein from the cell lysates used for immunoprecipitation were resolved by western blotting and probed with antibodies to phosphorylated ERK (pERK), total ERK, and β-tubulin (loading control). C, pERK and total ERK bands were quantified with Image Studio 2.0 (LI-COR Biosciences). Graphs depict the ratio of pERK to total ERK in the same sample, with vehicle-treated samples set as 1.0. Data represent means ± SD of three independent experiments. D, serum-starved cells were incubated with vehicle (−) or 40 μM CGS9343B (CGS, +). After 16 h, DMSO (−) or 100 ng/ml EGF (+) was added for 10 min. Samples were processed as described for panel A. All images are representative of three independent experiments. E, the calmodulin and KSR1 bands were quantified with Image Studio 2.0 (LI-COR Biosciences). Graphs depict the ratio of calmodulin (CaM) to KSR1 in the same sample. Data represent means ± SD of three independent experiments. F, serum-starved KSR1+/+ MEFs were incubated with vehicle (−) or 40 μM CGS9343B (CGS, +) for 16 h. Cells were lysed and 1 mg protein lysate was immunoprecipitated with anti-KSR1 antibody. A sample precipitated with mouse IgG was processed in parallel as negative control. Both unprocessed lysate (Input) and immunoprecipitates (IP) were resolved by western blotting and blots were probed with anti-KSR1 and anti-MEK1 antibodies. G, cells were treated as described for panel F, except the blots were probed with anti-KSR1 and anti-ERK antibodies. All data are representative of three independent experiments.