Fyn tyrosine phosphorylation at Y²¹⁵ and Y⁵²⁹ is markedly increased in anergic T cells. (A) Lysates of resting, anergic, and rescued T cells were probed with phosphospecific antibodies for either the activatory or the inhibitory tyrosine of Fyn, Y⁴¹⁸ and Y⁵²⁹, respectively. The level of tyrosine phosphorylation was normalized to total Fyn and is presented as the fold-induction. (B) The samples in panel A were probed with phosphospecific antibodies against the activatory and inhibitory tyrosines of Lck, Y³⁹⁴, and Y⁵⁰⁵ and normalized to total Lck. Black vertical lines have been inserted to indicate where these images were cut together from one film. (C) Cyclic AMP (cAMP) levels were determined. The data represent the mean (± SD) from 3 independent experiments. Data were analyzed using one-way ANOVA (∗∗, P < .01). (D) The level of Ser364 phosphorylation within Csk was determined by Western blotting. Total Csk staining is also shown. The level of phosphorylation normalized to total Csk is presented as the fold induction. (E) To investigate the mechanism responsible for increased Fyn kinase activity, Y²¹⁵ phosphorylation was investigated. Anti-pY²¹⁵ immunoprecipitates from resting, anergic, and rescued T cells were probed with anti-Fyn (top), anti-pY529 (middle), and anti-Lck (bottom). Because the IPs from anergic T cells contained more Fyn, we therefore concluded that phosphorylation of this site was enhanced in anergic cells.