Fig. 5. Activation kinetics of endogenous NFAT in Jurkat T cells. (A) Time-lapse images of GFP–NFAT translocation (upper panel) and samples of anti-NFAT4 immunofluorescence images (lower panel) obtained at the indicated time-points after Ca²⁺ stimulation. (B) Time courses of nuclear translocation of endogenous NFAT4 (circles) and GFP–NFAT (lines) upon Ca²⁺ stimulation. Experiments were carried out at room temperature (open circles, n = 9–12; continuous line, n = 7) and 37°C (filled circles, n = 11–17; dashed line, n = 6) (mean ± SEM). The ranges of errors are shown only at selected time points for clarity in the GFP–NFAT experiments. (C) Dephosphorylation of endogenous NFAT4 following Ca²⁺ stimulation. Whole-cell lysate prepared from Jurkat cells stimulated with Ca²⁺ at either room temperature or 37°C were used for western blotting with rabbit antiserum raised against NFAT4. Dephosphorylation of NFAT4 resulted in the mobility shift of the immunoblot bands.