Association of 4.1R with LAT and effect of 4.1R on LAT phosphorylation. (A) Association of 4.1R with LAT in situ. 4.1R was immunoprecipitated from 4.1R^+/+ or 4.1R^−/− T cells using anti-4.1R or preimmune IgG. 4.1R or LAT in the immunoprecipitate was detected using anti-4.1R antibody (i) or anti-LAT antibody (ii), respectively. (B) Crystal structure of membrane domain of 4.1R. Note the 3-lobe structure of the 30-kDa membrane binding domain. (C) Direct binding of 4.1R with LAT and inhibition of LAT phosphorylation by 4.1R in vitro. (i) Binding of 4.1R to cytoplasmic domain of LAT. 4.1R was incubated for 30 minutes at room temperature with MBP-tagged cytoplasmic domain of LAT, and binding was assessed by pull-down assay. 4.1R binding was detected by blotting with anti-4.1R exon 13 antibody after SDS-PAGE. (ii) Binding of cytoplasmic domain of LAT to recombinant 4.1R fragments. Recombinant His-tagged cytoplasmic domain of LAT was incubated with GST-tagged 4.1R fragments. Binding was assayed as above, using anti-His antibody for detection. (iii) Binding of cytoplasmic domain of LAT to subdomains of 4.1R 30-kDa membrane binding domain. Binding assays were performed as above. The minimum binding was mapped to lobe C of the 30-kDa domain. (D) Inhibition of in vitro LAT phosphorylation by 4.1R. Cytoplasmic domain of LAT was phosphorylated by ZAP-70 in the absence or presence of 4.1R or 4.1R fragments. The phosphorylation was detected by Western blot analysis with the use of anti-phosphotyrosine antibody 4G10. Note the phosphorylation was inhibited by 4.1R, 30 kDa of 4.1R, and lobe C of 30-kDa domain.