Fig. 9.

SHP-2–mediated regulation of the interaction among Lck, TCR, and ZAP-70. (A to C) Jurkat cells were transfected with empty vector, plasmid encoding SHP-2 (C/S), or plasmid encoding WT SHP-2 and stimulated by TCR cross-linking for 5 min as described for Fig. 3. TCRζ (A) or Lck (B and C) was immunoprecipitated from precleared lysates, and immunoprecipitates were analyzed by Western blotting with antibodies against ZAP-70, TCRζ, Lck, or SHP-2 or with antibody against phosphotyrosine to detect tyrosine phosphorylation of the TCRζ chain. (D) Human CD4⁺ T cell blasts were transfected with NT or SHP-2–specific siRNAs and stimulated by TCR cross-linking for 5 min. Post-nuclear cell lysates were analyzed by Western blotting with an antibody against SHP-2. Lck was immunoprecipitated and samples were analyzed by Western blotting with an antibody against pY319 ZAP-70. (E) CD4⁺ human T cell blasts were transfected with NT or Duox1-specific siRNAs and stimulated by TCR cross-linking as described for Fig. 6. Lck was immunoprecipitated from precleared lysates, and samples were analyzed by Western blotting with an antibody against pY319 ZAP-70. (F) Jurkat cells were transiently transfected with empty vector or with plasmid encoding HA-tagged Duox1, and cells were stimulated by TCR cross-linking as described for Fig. 3. Lck was immunoprecipitated from pre-cleared lysates, and samples were analyzed by Western blotting with an antibody against pY319 ZAP-70.