Fig. 6. Involvement of CaN in IgE-dependent NFAT2 nuclear translocation in human neutrophils. Neutrophils from an allergic patient were pretreated with CsA (A) or VIVIT peptide (B) at the indicated doses for 1 hour, and then treated with 10 µg/ml anti-IgE (-IgE) for 4 hours. Nuclear extracts were obtained, and NFAT2 levels analysed by western blotting, using anti-human NFAT2-specific Abs. Equal amounts of protein were loaded per lane. Histograms above each lane show the mean (± s.e.m.) values quantified from the blots from three separate experiments. (C) Whole-cell lysates (1 mg of protein) from untreated neutrophils from an allergic patient were incubated at 4°C for 18 hours with anti-CaN Abs. After immunoprecipitation (IP), the pellets were analysed by western blotting, using anti-NFAT2 (left upper panel). Thereafter, the blot was stripped, and reprobed with anti-CaN (left lower panel). Immunoprecipitation was carried out as indicated in A, except that anti-NFAT2 Abs was used, and subsequent western blotting analysis was carried out using anti-CaN Abs (right upper panel). After stripping, the blot was reprobed with anti-NFAT2 Abs (right lower panel). The signals directly obtained from neutrophil whole-cell lysates (80 µg) subjected to western blotting analysis without immunoprecipitation (WL) are also shown in both panels.