Figure 4.

AHNAK1 is required for calcium signalling and NFAT activation upon TCR stimulation. (A) CD4 T cells from wild-type and AHNAK1^−/− mice were purified and stimulated in vitro using plate bound anti-CD3 (10 μg/ml), anti-CD28 (2 μg/ml) and exogenous IL-2 for 48 hrs. Cells were then washed and incubated with anti-CD3 (10 μg/ml) for 30 min on ice and were subsequently cross-linked by goat anti-hamster Ig antibody (GAH). Calcium concentration was measured by ratiometric method using Fura-2 as a probe. Results are representative of at least three independent experiments. (B) CD4 T cells were stimulated with anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml) antibodies for the indicated periods. Nuclear localization of NFATc1 and NFATc2 was examined by western blot analysis using cytoplasmic or nuclear extracts. β-actin was used as internal control. Results are representative of two independent experiments, each with 10 wild-type or AHNAK1^−/− mice. (C) DNA binding of NFAT-c2 was examined using nuclear extracts from wild-type and AHNAK^−/− purified CD4 T cells. (D) [³H] thymidine incorporation assay was performed after stimulation of CD4 cells with plate bound anti-CD3 antibody (2 μg/ml) with or without addition of ionomycin (0.5 μM) into the culture. Results are representative of at least three independent experiments