Figure 3

Differential Lck phosphorylation of variant TSAd molecules. A. Tyrosine phosphorylation of TSAd variants in Jurkat T cells: Jurkat T cells transiently transfected with pEF-HA or pEF-HA-SH2D2A-1-5 cDNAs, were stimulated (+) with anti-CD3 (OKT3) mAbs for 2.5 min or left unstimulated (-), lysed and subjected to immunoprecipitation with anti-TSAd Abs (TSAd IP). The precipitates were separated by SDS-PAGE and immunoblotted with anti-phoshotyrosine (pY) mAbs (upper panel) and anti-HA mAbs (lower panel). Relative level of tyrosine phosphorylation of TSAd variants is shown in the chart, and the relationships between the pY signal versus the HA signal of bands is normalised to that observed for the full length TSAd IP (SH2D2A-1) expressed in resting Jurkat T cells. B. Lck phosphorylates the SH2D2A-1, -2 and -3 variants of TSAd in 293T cells: 293T cells were transiently transfected with pEF-HA or pEF-HA-SH2D2A-1-5 cDNAs alone (-) or together (+) with pEF-Lck. The cells were lysed and treated as in A. C. Tyrosine phosphorylation of TSAd mutated for the four C-terminal tyrosines is attenuated when co-expressed with Lck in 293T cells. 293T cells were transiently transfected with pEF-HA, the pEF-HA-TSAd-4YF or pEF-HA-TSAd-d239–256 cDNAs together with pEF-Lck. The cells were lysed and treated as in A.