ATM phosphorylation is stimulated on T-cell activation with anti-CD3, PMA/ionomycin or SEB antigen. (A) PBMCs were starved for 18 hours in 0.1% fetal calf serum, and then left untreated or stimulated with PMA (50 ng/mL) plus ionomycin (500 ng/mL), or with anti-CD3 mAb followed by a GAM cross-linking, for the indicated times at 37°C. Cell lysates (100 μg/lane) were immunoblotted with anti-phospho-ATM (specific for the phosphoserine 1981), or with anti-phospho-JNK/SAPK, used as a control of T-cell activation. Protein loading was normalized after stripping and reprobing the latter membrane with antitotal-JNK/SAPK mAb, or with an antipaxillin mAb. Data shown derive from the same experiment and are representative of 1 of 2 (for CD3) or 3 (for PMA/ionomycin) independent experiments. (B) Densitometric analysis of the bands shown in panel A, and relative to pATM (upper and lower bands) and to paxillin (as a control). The relative protein level of stimulated samples with respect to that of unstimulated cells (time 0) is shown. AU, arbitrary units. (C) PBMCs were starved and then stimulated with PMA/ionomycin or anti-CD3⁺GAM as described in panel A, or incubated with VP16 (10 μM) for 2 hours, and lysed immediately after. Data derive from the same donor analyzed in the same experiment and are representative of 2 donors. (D) PBMCs were starved and stimulated as described, and then stained anti-pATM-Ser1981 (thick line) or cIgG (filled histogram). MFI values relative to pATM are shown. Data are from 2 different donors, one of which (SEB/VP16) is the same as in panel C.