Fig. 4. Differences in phosphotyrosine signaling by the ß1-787-789 (^*/A) and ß3-751-753 (^*/A) mutants. (A) Normal human fibroblasts expressing tac chimeras containing either the wild-type ß1 or ß3 cytoplasmic domain or the ß1-787-789 (^*/A) or ß3-751-753 (^*/A) mutant were incubated in clustering assays for 40 minutes or 90 minutes. Lysates (10 µg/sample) were analyzed by SDS-PAGE and blotted for phosphotyrosine (upper panel), and then reprobed for p130CAS (lower panel). Samples were loaded as indicated. (B) Samples from the total population of transfected cells used for the study in A were examined by flow cytometry for the surface expression of the tac subunit of the IL-2 receptor. (C,D) Human fibroblasts were transfected with tac chimeras containing either the wild-type or mutant tails as indicated. Tac chimeras were clustered on the cell surface for 40 minutes. After preparation of cell lysates, p130CAS was immunoprecipitated from 200 µg of protein in C, or paxillin was immunoprecipitated from 150 µg in D and then separated by SDS-PAGE, blotted for phosphotyrosine (upper panel) and reprobed for p130CAS or paxillin (lower panels). Samples were loaded as indicated above each lane. These experiments were performed twice and similar results were obtained. P130, p130CAS; pax, paxillin.