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. 2021 Apr 16;296:100674. doi: 10.1016/j.jbc.2021.100674

Figure 7.

Figure 7

Translocation of WT, K49E, and I51C GFP-mSK1 in MCF-7L cells. MCF-7L cells overexpressing WT GFP-mSK1 or K49E GFP-mSK1 or I51C GFP-mSK1 were treated with S1P (5 μM) or PMA (1 μM) or carbachol (100 μM) for 10 min. Cells were processed and mounted with DAPI to stain the DNA (blue). A, photomicrographs of GFP fluorescence in cells of 40× oil magnification overexpressing WT, K49E or I51C GFP-mSK1. Results are representative of three independent experiments. Inset is a Western blot probed with the anti-GFP antibody showing the overexpression of GFP-mSK1, and the bar graph shows transfection efficiency (%). No significant difference between WT versus mutants (one-way ANOVA with Tukey's post hoc test). B, the bar graphs represent the AUC (right) of transfected GFP-mSK1 (WT or mutant) translocation (n = 5) and the percentage of cells (left) containing translocated GFP-mSK1 (WT or mutant) (n = 3); ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 for K49E or I51C mutant versus WT for a given stimulus; +p < 0.05, ++p < 0.01, and ++++p < 0.0001 for stimulus versus respective control for transfected WT GFP-mSK1 or K49E or I51C mutants (two-way ANOVA with Tukey's post hoc test). C, bar graphs representing the percentage of cells containing GFP-mSK1 (WT or K49E or I51C) in the microdomains in the PM of lamellipodia and filopodia in response to stimulus (n = 4); ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 for stimulus versus control for transfected WT GFP-mSK1 or K49E or I51C mutants (two-way ANOVA with Tukey's post hoc test). AUC, area under the curve; mSK1, mouse SK1; PMA, phorbol 12-myristate 13-acetate; S1P, sphingosine-1-phosphate.