FIG. 9.

Binding of phosphotyrosyl proteins to the PLC-γ1 SH2(C) domain. (A) Precipitations with GST fusion proteins. P98 cells were stimulated for 2 min with medium only or 100 μM PV. Detergent extracts were precipitated with 2.5 μg of immobilized GST only or GST-γ1 SH2(C) fusion protein. The bound proteins were separated by SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody. The protein bands indicated with arrows were identified as SLP-76 and ZAP-70 by subsequent immunoblotting of the same membrane with the respective protein-specific antisera. The numbers on the left indicate molecular weights of protein calibration markers. (B) SH2(C) domain-dependent binding of SLP-76. P98 cells were transiently transfected with pcaPLC-γ1 WT (5 μg), SH2(N)* (5 μg), or SH2(C)* (15 μg) plasmid DNA plus either pcDNA3 or FLAG-tagged SLP-76-encoding plasmid DNA (5 μg). Samples were stimulated for 5 min with either medium only or 100 μM PV. Detergent-soluble proteins were immunoprecipitated (IP) with an anti-AU1 MAb, separated by SDS-PAGE, and immunoblotted with an anti-FLAG MAb. The membrane was stripped and reprobed with the anti-AU1 MAb to determine the amount of PLC-γ1 immunoprecipitated in each sample. An aliquot of the whole-cell extract (WCE) was immunoblotted with the anti-FLAG antibody to determine the expression level of FLAG–SLP-76 in each sample (bottom).