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

Figure 6.

Figure 6

Inhibiting calmodulin attenuates EGF-induced translocation of KSR1 to the plasma membrane and its association with ERK and pERK.A, serum-starved KSR1+/+ MEFs were pretreated with DMSO (V) or 40 μM CGS9343B (CGS). After 16 h, cells were incubated without (−) or with (+) 100 ng/ml EGF for 5 min. Cells were fixed, then probed with anti-KSR1 and anti-Na+K+ ATPase antibodies. Following incubation with the appropriate fluorescent-conjugated secondary antibodies, images were analyzed by Zeiss LSM780 confocal microscopy. The smaller panels on the right side are higher power magnifications of the areas encompassed by the white boxes on the images on the left. Merge is a composite of both channels; KSR1 is green, Na+K+ ATPase is red, and yellow indicates colocalization. Scale bars: full panels, 10 μm; insets, 3 μm. The data are representative of 50 cells for each condition. Negative controls showed no evidence of nonspecific staining or bleed-through. B, Pearson’s correlation coefficient was determined for colocalization of KSR1 and Na+K+ ATPase with Zen software. The data are expressed as means ± SD (n = 50 cells), with vehicle-treated cells set as 1. ∗p < 0.0001 using one-way ANOVA, with Tukey’s post-hoc test. C and E, KSR1+/+ MEFs were treated as described for panel A, then stained with both anti-KSR1 and anti-pERK (C) or anti-KSR1 and anti-ERK (E) antibodies. PLA was performed using Duolink detection reagents as described under Experimental procedures. Red spots indicate positive PLA. Actin was stained with Alexa Fluor 488 phalloidin (green). Representative images of 50 cells for each condition are shown. The smaller panels on the right side are higher power magnifications of the areas encompassed by the white boxes on the images on the left. Scale bar, full panels, 10 μm; insets, 3 μ. D and F, the number of PLA spots per cell was quantified with Image J software from confocal images of 50 cells for each condition. The data are expressed as means ± S.D. (error bars), with the number of spots in vehicle-treated cells set to 1. ∗p < 0.0001 by one-way ANOVA, with Tukey’s post-hoc test.