Figure 3.

K1 requires syk for high-level signal transduction and induces phosphorylation of syk and PLCγ2. (A) Histograms of the fold activation of luciferase in DT-40 syk negative cells cotransfected with the indicated plasmids and the NFAT-luc plasmid, with or without an expression plasmid containing human syk, versus activity in cells transfected with the empty vector. (B) An antiphosphotyrosine (4G10) Western blot of Raji cells transfected with the indicated plasmid and a syk expression construct and precipitated with a human syk antibody. The right side is the same blot stripped and reprobed with an anti-syk antibody. (C) A Western blot of Raji cells transfected with the indicated plasmid, immunoprecipitated with 4G10, and blotted first with an anti-syk antibody followed by an anti-HA antibody. (D) Anti-syk Western blot of Raji cell lysates incubated with the indicated unphosphorylated, singly phosphorylated, or doubly phosphorylated biotin-labeled peptide and precipitated with avidin-agarose. (E) Anti-PLCγ2 Western blot of Raji cells transfected with the indicated plasmid and immunoprecipitated with 4G10. The right side contains the supernatant from the immunoprecipitates.