Fig. 3.

NCS-1 and NCS-1 mutant proteins do not affect dephosphorylation of NFAT-GFP over an acute time course of intracellular Ca²⁺ elevation. (a) Anti-GFP western blots on HeLa cell lysates from cells transfected with NFAT-GFP and the indicated pcDNA based expression constructs. Cells were treated with Ca²⁺/ionomycin for the indicated times and then lysed directly into SDS dissociation buffer, separated on SDS-PAGE (12.5% gel) and transferred to nitrocellulose for western blotting with an anti-GFP antibody. Fully phosphorylated NFAT-GFP (P, upper arrow) present at time 0 for all transfection conditions was dephosphorylated on Ca²⁺/ionomycin treatment as monitored by the appearance of more rapidly migrating species by the 5 min time point for all transfection conditions (De-P, lower arrow). (b) Anti-calcineurin western blot on samples derived from an in vitro binding assay where recombinant CN (1 μM) was incubated with GST control protein, GST-NCS-1 or GST-NCS-1^E120Q (all at 1 μM) in the absence (-Ca²⁺) or presence (+Ca²⁺) of 1 μM free Ca²⁺. Samples from duplicate incubations are presented.