FIGURE 4:

STIM1-ORAI1 puncta-like formation is impaired in KSR2-depleted HeLa cells. (A–C) ShRNA control and shRNA KSR2 HeLa cells were grown on coverslips and cotransfected with expression vectors encoding YFP-STIM1 (0.2 μg) and ORAI1-RFP (0.2 μg). After starvation for 2 h, cells were stimulated either without (basal) or with 1 μM Tg. Distribution of Tg-induced STIM1-ORAI1 puncta formation (yellow dots) was visualized under confocal microscopy. Bar, 5 μm. (B–D) shRNA KSR2–depleted HeLa cells were cotransfected with expression vectors encoding YFP-STIM1 (0.2 μg) and ORAI1-RFP (0.2 μg) and Flag alone 0.2 μg (B) or KSR2-Flag 0.2 μg (D). After starvation, cells were stimulated as in A–C. Cells were fixed, permeabilized, and incubated with anti-Flag antibody, followed by Alexa Fluor 647–labeled secondary antibody. Tg-induced STIM1-ORAI1 puncta formation (yellow dots) was visualized under confocal microscopy. Bar, 5 μm. One representative cell of >30 cells in three experiments. White squares show enlarged images. Bar, 1 μm. The graph bars illustrate the mean per cell ± SEM of puncta in response to Tg treatment in >30 cells/condition. Significance of values is compared with control Tg-treated cells. *p < 0.05.