Fig. 2.

Both the kinase domain and death domain of DAPK1 are required for mTORC1 activation. WT and KD-deficient CTLs were starved overnight with serum-free RPMI 1640 overnight and then stimulated with anti-CD3/CD28 (2 μg/ml of each) (a, b) or rhIL-2 (20 ng/ml) (c, d) for the indicated times. Expression of phosphorylated forms and total proteins of p70S6K, S6, AKT-T308 and -S473, NFAT, p65, Erk1/2, and p38 were determined by immunoblot analysis. PLC-γ and STAT5 activation were measured in a and c, respectively. Representative graphs are shown in a and c, and the band intensities were quantified and analyzed for statistical significance in b and d. To detect NFAT phosphorylation, cytosolic fractions were isolated using a cytosolic/nuclear fractionation kit, and NFAT phosphorylation was determined by immunoblot. WT and DD-deficient CTLs were starved with serum-free RPMI 1640 media and then stimulated with anti-CD3/CD28 (2 μg/ml of each) (e, f) or rhIL-2 (20 ng/ml) (g, h) for the indicated times. Immunoblot analysis was performed as in a–d