PAG down-regulation enhanced both Src kinase activity and Ras activation. Primary human T cells (A) and Jurkat T cells (B) were transfected with siRNA oligonucleotides or the plasmid pCMS3-EGFP containing PAG shRNA, respectively. The down-regulation of PAG protein expression compared with the control is shown. Cells were stimulated with CD3 and CD28 for the times indicated, and lysates blotted with anti-phosphotyrosine. Actin staining is included to show equal loading. (C) In addition, the ability of transfected Jurkat cells to activate Ras was determined using the GST-Raf1-RBG pull-down assay. Total Ras is included as the control and the normalization of active/total Ras appears in the graph. Values represent the mean (± SD) of 4 independent experiments. Data were analyzed using the Student t test (∗, P < .05; ∗∗, P < .01). The down-regulation of PAG protein expression is shown.