Figure 5.

ZAP70 requires co-expression of an ITAM and Src-family kinases activity for activation. (A) S2 cells were co-transfected with the indicated proteins and pretreated with SykIV inhibitor (2.5 µM), SYKII inhibitor (2.5 µM), PP1 (10 µM), PP2 (10 µM), or DMSO solvent control 1 h before harvesting. Equal numbers of FACS-enriched GFP⁺ cells were lysed and analyzed by western blot for SLP-76 phosphorylation and expression of transfected proteins. All western blots are representative of at least three independent experiments. (B) Quantification of SLP-76 Y128 phosphorylation relative to total SLP-76 expression upon co-expression of CD3ζ, Fyn, SYK, and SLP-76 after pretreatment with the indicated inhibitors or DMSO control. (C) Quantification of SLP-76 Y128 phosphorylation relative to total SLP-76 expression upon co-expression of CD3ζ, Fyn, ZAP70, and SLP-76 after pretreatment with the indicated inhibitors or DMSO control.