Fig. 3.

Impaired TCR signaling in peripheral T cells from Lck^Y192E knock-in mice. a Splenic T cells from Lck^wt and Lck^Y192E knock-in mice were stimulated with a CD3 antibody. At the indicated time points after stimulation, cells were lysed and the levels of global protein tyrosine phosphorylation were assessed using a pan phosphotyrosine antibody (pY total). One representative experiment is shown (n = 4). b The activation/phosphorylation of selected and important signaling molecules in T cells was investigated using the indicated phosphospecific antibodies. Equal protein loading was verified using antibodies directed against corresponding total proteins or against β-actin, respectively. c Calcium influx in peripheral T cells was assessed upon CD3xCD4 cross-linking by flow cytometry. The Ca²⁺ flux was calculated via the ratio of Indo-1 high/Indo-1 low expressing cells. Ionomycin was used to show equal loading with Indo-1. One representative of three independent experiments is shown. d Proliferation of splenic T cells was assessed using [3H]-thymidine incorporation upon stimulation with a CD3 antibody (1 µg). PMA + ionomycin were used as positive control. One representative of four independent experiments is shown