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

Figure 5.

Figure 5

Conditional changes in protein interaction detection between WT Myc-mSK1 and WT GFP-mSK1 using Duolink Proximity Ligation Assay (PLA).A, overexpression of WT Myc-mSK1 or WT GFP-mSK1 was confirmed in MCF-7L cells by fluorescence microscopy before Duolink PLA to assess protein interaction. PLA was carried out to assess differences in protein interaction between WT Myc-mSK1 and WT GFP-mSK1 (unstimulated control) and upon treatment with either S1P (5 μM) or PMA (1 μM) or carbachol (100 μM) for 10 min. Untransfected cells (No-TF) and cells expressing only WT GFP-mSK1 or WT Myc-mSK1 were used as negative controls. Cells were processed for PLA and mounted with DAPI to stain the DNA (blue). B, quantitative analysis of the cellular PLA signals was carried out. The bar graphs represent the PLA signal mean gray value (mean ± SEM) with experiments carried out in triplicate (30 cells total per treatment group shown, n = 3) ∗∗p < 0.01 and ∗∗∗p < 0.001 for stimulated versus control in WT Myc-mSK1/WT GFP-mSK1 transfected cells (one-way ANOVA with Bonferroni post hoc test). C, representative PLA images are shown for each treatment group. The scale bar represents 20 μm. Results are representative of three independent experiments. mSK1, mouse SK1; PMA, phorbol 12-myristate 13-acetate; S1P, sphingosine-1-phosphate.