PAG overexpression and hyperphosphorylation lead to the block in Ras activation. Jurkat T cells were transfected with constructs encoding for either PAG, Y317F, Fyn, LAT, and/or vector alone as indicated. The following day, cells were stimulated with anti-CD3 and anti-CD28 for the indicated times, lysed, and active Ras (ie Ras-GTP) isolated using the Raf1-RBD (A-C, E, and F, left panels). Total Ras is shown to prove that equal amounts of material were used for this assay (A-C, E, and F, right panels). Representative experiments are shown. A graphical representation of the data is also presented. Values represent the mean (± SD) of at least 3 independent experiments. Data were analyzed using one-way ANOVA (∗, P < .05). Changes in protein expression and phosphorylation after transfection are presented using immunoblots from lysates probed with the indicated antibodies (Figure S2). (D) Jurkat T cells were transfected with constructs encoding for either PAG, the individual YΔF mutants, or vector alone. RasGAP was then immunoprecipitated and the blots probed for associated PAG (anti-FLAG) staining and RasGAP. Blots of the transfected cell lysates are also shown. A black line has been inserted to indicate that, due to space limitations, the samples were resolved on 2 gels.