Fig. 4. The KIR- and SH2-domains of SOCS proteins participate in SOCS–FAK interaction. (A and B) COS-7 cells were transiently transfected with indicated plasmids (0.2 µg of HA-FAK or HA-JAK2, 0.5 µg of Myc-SOCS-1/3). Forty-eight hours after transfection, cell lysates were immunoprecipitated with anti-HA antibody, and the precipitates were analyzed by immunoblotting with antibodies against Myc or HA. Total cell lysates were subjected to immunoblot with anti-Myc antibody to confirm SOCS protein expression levels. The relative FAK-SOCS interaction was determined by a densitometric analysis and the NIH Image 1.62 program. The KIR-mutant form of SOCS-1 (SOCS-1-F59EdN) and the SOCS-1 double-mutant (SOCS-1-F59E, R105K) contain a deletion of the first 51 amino acids that has been shown to stabilize the expressed protein without changing its binding affinity or specificity (Yasukawa et al., 1999).