Figure 4

Tyrosine phosphorylation of CD300a requires the src kinase Lck. (A) KIR-CD300a WT and KIR-CD300a 4F Jurkat T cells were stimulated with medium or pervanadate for 3 minutes, or mixed with 721.221-Cw3 or 721.221-Cw6 and incubated at 37°C for 5 minutes. Cell lysates were immunopreciprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for phosphotyrosine and HA. Results are representative of five independent experiments. (B) E6.1 Jurkat cells, Jurkat cells deficient in Lck or deficient in ZAP-70 were transiently transfected with a plasmid encoding KIR-CD300a and incubated at 37°C with 721.221-Cw3 and 721.221-Cw6 for 5 minutes. Cell lysates were immunoprecipitated with anti-KIR2DL2 (clone GL183) mAb and blotted separately for phosphotyrosine and HA. Results are representative of three independent experiments.