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. 1998 Apr 20;187(8):1249–1260. doi: 10.1084/jem.187.8.1249

Figure 7.

Figure 7

Figure 7

Distinct patterns of tyrosine phosphorylation are induced in immature F5/RAG-1−β2m− thymocytes by stimulation with CD3/CD4 BsAb and CD3fos-F(ab′)2. (A) Thymocytes were stimulated with F(ab′)2 reagents, as indicated, for 8 min at 37°C. Lysates were immunoprecipitated with anti–TCR-ζ mAb and the precipitates immunoblotted with antiphosphotyrosine mAb (4G10), to reveal differences in TCR-associated phosphotyrosine-containing proteins. (B) Total ZAP-70 proteins associated with TCR-ζ polypeptides were examined in unstimulated or F(ab′)2-stimulated samples (5 min at 37°C), as indicated. Lysates were immunoprecipitated with anti–TCR-ζ and immunoblotted with anti–ZAP-70 mAb. (C) Tyrosine phosphorylation of ZAP-70 proteins in control or F(ab′)2-stimulated thymocytes was visualized by immunoprecipitating cell lysates with polyclonal anti–ZAP-70 serum after stimulation of thymocytes for 5 and 20 min at 37°C, as indicated, and immunoblotting was with antiphosphotyrosine 4G10. Half of the 20-min precipitate was probed with anti–ZAP-70 mAb to confirm that equivalent amounts of protein were present. (D) p56lck immunoprecipitates demonstrate different association with TCR-associated phosphoproteins. Thymocytes were stimulated with F(ab′)2-reagents at 37°C for 5 min, as indicated, or left on ice throughout (4°C). Cell lysates were precipitated with p56lck-antiserum and immunoblotted with antiphosphotyrosine (4G10).