Figure 4

SH2D2A exon 7 encodes ligands for Lck-SH2 and SH3 domains. A. TSAd interacts with Lck: Primary CD4+ T cells were stimulated with anti-CD3/CD28 beads for one day. TSAd was precipitated with anti-TSAd Abs or irrelevant serum (NRS) and protein A/G sepharose beads from precleared lysates. Precipitates were separated by SDS-PAGE and immunoblotted with anti-Lck and anti-TSAd Abs as indicated. B. TSAd variants interact with Lck in Jurkat T cells: Jurkat T cells were transiently transfected with pEF-HA or one of the pEF-HA-SH2D2A-1-5cDNAs. Immunoprecipitation was performed using anti-TSAd Abs and protein G magnetic beads. Precipitates were separated by SDS-PAGE and immunoblotted with anti-Lck and anti-HA as indicated. C. The SH2 domain and aa239–334 of TSAd is important for interaction with the Lck SH2 domain: 293T cells were transiently transfected with one of the pEF-HA-SH2D2A-1-5 cDNAs alone (-) or together (+) with pEF-Lck. Cell lysates were subjected to pull down experiment with GST-Lck-SH2 Sepharose beads. Precipitates were immunoblotted with anti-HA mAbs (upper panel). An anti-HA immunoblot of precleared lysates before GST-Lck-SH2 pull down is included to verify expression of the different HA-tagged TSAd variants (lower panel). D. Aa239–334 contains ligands for the Lck-SH3 domain: 293T cells were transiently transfected with pEF-HA or one of the pEF-HA-SH2D2A-1-5 cDNAs together (+) with pEF-Lck. Only the pEF-HA-SH2D2A-1 cDNA were also co-transfected with pEF-HA (-). Cell lysates were subjected to pull down experiment with GST-Lck-SH3 Sepharose beads, and precipitates (upper panel) and precleared lysates (lower panel) were immunoblotted as in C. E. Identification of TSAd structures interacting with Lck domains: 293T cells were transiently transfected with pEF-HA, the pEF-HA-TSAd-4YF or pEF-HA-TSAd-d239–256 cDNAs together with pEF-Lck. Cell lysates were subjected to pull down experiment with GST-Lck-SH2 or GST-Lck-SH3 Sepharose beads. GST-Lck-SH2 (panel 1) and GST-Lck-SH3 (panel 2) precipitates were immunoblotted with anti-HA mAbs, and the precleared lysates were immunoblotted with anti-HA mAbs (panel 3) or anti-Lck mAbs (panel 4). F. The SH2 domain of TSAd precipitates Lck in 293T cells: 293T cells were transiently transfected with pEF-HA, pEF-Lck, pEF-HA-SH2D2A-1 or pEF-Lck and pEF-HA-SH2D2A-1 cDNAs together. Precleared cell lysates were subjected to pull down experiment with GST-TSAd-SH2 Sepharose beads. Precipitates were immunoblotted with anti-Lck mAbs (upper panel). An anti-Lck immunoblot of precleared lysates before GST-TSAd-SH2 pull down is included to verify expression of Lck (lower panel).