Ly49A-mediated inhibitory signals diverge and converge with the CTLA-4 signaling pathway.

(A) Total tyrosine phosphorylation in lysates of purified lymph node T cells ex vivo. Lysates were equalized for total protein content and the proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 8%; lane 1: Ly49ATg⁺D^d CTLA-4^+/+,+/−; lane 2: Ly49ATg⁺D^d CTLA-4^−/−; lane 3: Ly49ATg⁺D^b CTLA-4^−/−). The phosphoproteins were detected by Western blotting using antiphosphotyrosine antibody 4G10. These data are representative of 7 independent experiments. Asterisks indicate bands uniquely phosphorylated in the CTLA-4^−/− T cells from mice that develop lymphoproliferation but not in the “rescued” (Ly49ATg^+/− D^d) CTLA-4^−/− or in the littermate control animals. (B) Phosphorylated ERK2 was detected by Western blot total protein with anti-EK2 tyrosine phosphorylation–specific antibody. The blots in panels A and B were stripped and reprobed with anti-ERK2 antibody (lanes are as indicated in panel A ). This is representative of 3 independent experiments. (C) CD3ζ phosphorylation. Lysates of equivalent total protein were immunoprecipitated by anti-CD3ζ antibody 387. Immunoprecipitates (lanes are as indicated in panel A) were separated by SDS-PAGE (12%) and phosphoproteins were detected by Western blotting using antiphosphotyrosine antibody 4G10. The blots were stripped and reprobed with anti-CD3ζ antibody. This is representative of 3 independent experiments.