Figure 5

Lck phosphorylation correlates with decreased T-cell activation. A) Jurkat T cells were transfected with Lck siRNA duplex or siRNA control (Ctrl) and cultured for 48 h. Subsequently, cells were stimulated with soluble CD3 mAbs (clone OKT3) for the indicated times. Cell lysates were analyzed by immunoblotting using the indicated Abs. Immunoblot verifying Lck downregulation is shown. One representative experiment of three independent experiments is shown. B)-D) Purified human CD4⁺ T cells were treated with immobilized (iAbs) CD3xCD28 mAbs in the presence or absence of cross-linked CD4 mAb as indicated. B) The phosphorylation levels of Erk1/2 and Src kinases were determined by Western blotting. The phospho-specific bands were quantified using the 1D ImageQuant software and the values were normalized to the corresponding β-actin signal. Data on the graph represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of 3 independent experiments. C) 24h after stimulation, the activation of CD4⁺ T cells was analyzed by staining with CD69 and flow cytometry. Data on the graph represent the mean of the expression levels shown as arbitrary units ± SEM of 3 independent experiments. D) CD4⁺ T cells were labeled with CFSE and stimulated as indicated. Proliferation was assed after 72h by analyzing CFSE content on a LSRFortessa. One representative experiment of 3 independent experiments is shown.