PAG is hyperphosphorylated in anergic T cells. (A) Anergic cells showed increased PAG-associated Fyn-kinase activity. PAG was immunoprecipitated from resting (rest.), anergic (aner.), and rescued (resc.) cells, and the PAG-associated kinase activity was measured by IVK. Phosphorylation was visualized by autoradiography (top panel). The amount of precipitated PAG and coprecipitated Fyn were detected by Western blotting (middle and bottom panels). Bands corresponding to PAG and enolase phosphorylation are marked. Phosphorylation of enolase was normalized with respect to the PAG level and is presented here as fold induction. (B) PAG was hyperphosphorylated in anergic cells. Resting, anergic, and rescued T cells were either left untreated (−) or stimulated via CD3 (MEM-92) for 2 minutes (+). Samples were then lysed, subjected to Western blotting, and probed with a phosphospecific antibody recognizing pY³¹⁷ of PAG (top panel). Actin staining is shown for equal loading (bottom panel). The level of Y³¹⁷ phosphorylation normalized to actin is presented as the fold-induction. (C) Hyperphosphorylated PAG recruits more Csk in anergic cells. Resting, anergic, and rescued cells were prepared as in panel B. PAG was immunoprecipitated and the associated proteins detected by blotting with anti-Csk and anti-Fyn antibody. The amount of Csk coprecipitated with PAG was normalized to the PAG level and is presented as fold induction.