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. 2021 Mar 23;296:100577. doi: 10.1016/j.jbc.2021.100577

Figure 5.

Figure 5

CGS9343B prevents EGF from activating ERK at the plasma membrane. Serum-starved KSR1+/+ MEFs (panels A and B), parental MEFs (C and D), and KSR1−/− MEFs (E and F) were pretreated with DMSO (−) or 40 μM CGS9343 (CGS, +) for 16 h, followed by incubation with vehicle (−) or 100 ng/ml EGF (+) for 5 min. Cell lysates were separated into cytoplasmic and membrane fractions as described under Experimental procedures. In total, 10 μl of whole-cell lysate (WCL; panel A, only), cytoplasmic and membrane fractions were resolved by SDS-PAGE and transferred to PVDF membranes. Blots were probed with anti-pERK, anti-ERK, anti-KSR1, anti-β-tubulin, and anti-Na+K+ ATPase antibodies. Na+K+ ATPase is the control for membrane fractions. Data in panels A and C are representative of three independent experiments and data in panel E are from a single experiment. B, D, and F, phosphorylation of ERK was quantified by densitometry using Image Studio 2.0 (LI-COR) and corrected for the amount of total ERK in the same sample. Data represent the means ± SD (panels B and D, n = 3 independent experiments) or a single value (panel F), with vehicle-treated cells set as 1.0. ∗p < 0.001 using one-way ANOVA, with Tukey’s post-hoc test.