Fig. 4

TSAd promotes Nck and Lck interaction. a Nck co-immunoprecipitation of TSAd and Lck protein expressed in 293T cells. 293T cells transfected with cDNA encoding Nck-GFP, HA-tagged TSAd and Lck were subjected to Nck-IP, and immunoblotted with the indicated antibodies. Data is one representative of three experiments. b and c Endogenous TSAd co-precipitate with Nck. Immunoblot of Nck-IP from JTAg cell lysates with TSAd expression (siRNA Control (Ctrl)) or TSAd knock down (siRNA TSAd (TSAd)). Cells were PMA/Io treated (20 h), and subsequently stimulated with anti-CD3 (1 min) or left unstimulated. Lysates were immunoblotted with the indicated antibodies. Results are one representative of three independent experiments. c Graph shows relative amounts of TSAd co-precipitated with Nck-IP as shown in (b) measured by densitometry (ImageJ). Mean values ± SD of three independent experiments (2-tailed paired t-test). d and e Co-precipitation of Lck with Nck. Cells were treated as described in (b). Amount Lck co-precipitated with Nck in unstimulated TSAd expressing cells (siRNA Control) was set to 1. KD: knock down. Mean values ± SD of three independent experiments (2-tailed paired t-test). ns: not statistically significant. f Nck-IP and lysates from resting CD4+ murine SH2D2A ^−/+ or SH2D2A ^−/− blast T cells. Results shown are one representative out of three independent experiments. g Graph shows relative amount of Lck co-precipitated with Nck IP as shown in (f) as measured by ImageJ. Amount of Lck co-precipitated with Nck in SH2D2A ^−/+ CD4+ T cells was set to 1. Mean values ± SD of three experiments. P-values are indicated (2-tailed paired t-test)