Fig. 2.

Over-expression of NCS-1 and NCS-1 mutant proteins has no detectable effect on NFAT dephosphorylation or NFAT nuclear translocation. (a) Anti-GFP western blot analysis of samples from HeLa cells co-transfected with an NFAT-GFP reporter construct in addition to control empty vector (pcDNA) or the indicated pcDNA based NCS-1 expression constructs. Cells were treated with Ca²⁺/ionomycin for the indicated times and lysed for SDS-PAGE analysis/western blotting with anti-GFP antibody. Fully phosphorylated NFAT-GFP was detectable in all transfection conditions at the zero time point (P, upper arrow) and dephosphorylation of this reporter was clearly observable after 7 mins of Ca²⁺/ ionomycin treatment with a gel shift to two more rapidly migrating species corresponding to dephosphorylated forms of the protein (DeP, lower arrows). (b) Confocal images of NFAT-GFP fluorescence from HeLa cells transfected and treated as for (a) but subsequently fixed and processed for microscopy. NFAT-GFP fluorescence was diffusely cytosolic and excluded from cell nuclei under control, zero time point, conditions for all transfected constructs tested. On elevation of cytosolic Ca²⁺ for 7 mins (ionomycin) NFAT-GFP fluorescence was observed to translocate from the cytosol to cell nuclei for all transfected constructs analysed, consistent with dephosphorylation of the protein observed in parallel western blot studies (a). Scale bars 10 μm. (c) Western blot of total HeLa cell lysates with an anti-NCS-1 antiserum. Cells transfected identically to those used in functional assays (a and b) were lysed and subjected to western blotting to determine relative over-expression levels of the NCS-1 proteins analysed in this part of the study. Essentially equivalent levels of expression were detected for NCS-1, NCS-1G2A and NCS-1E120Q constructs. No NCS-1 signal was detectable in control empty vector transfected samples (pcDNA).