Fig. 2.

The proline rich region (PRR) of TSAd interacts with the Nck SH3 domains. a GST-Nck SH3 pull-downs from 293T cells transiently expressing the indicated HA-tagged TSAd constructs. Proteins were resolved on SDS-PAGE, and immunoblotted with the indicated antibodies. Control GST pull-downs were negative (data not shown). Lck immunblot of the GST-Nck SH3 pulldown was negative (data not shown). Results shown are from the same experiment as shown in Fig. 1e and are representative of at least three experiments. b Peptide spot array spotted with proline containing TSAd peptides probed with GST-Nck SH3 peptides and developed using anti-GST antibody. Nck SH3 domain interaction sequences on TSAd are boxed in grey, the TSAd region aa 239–274 is outlined and canonical SH3 ligand motifs and the core PXXP motifs are underlined with black or red lines respectively. c Pull-down with single GST-Nck SH3 domains and control GST performed as in (a). Results are one representative of three independent experiments. d Pull-down of GST-Nck SH3 domains in JTAg cells transiently expressing HA-TSAd WT. Positive controls: Itk SH3 and Lck SH3. Negative control: GST. Blots were immunoblotted with the indicated antibodies. Data is one representative of two experiments. e Nck co-immunoprecipitation of TSAd expressed in 293T cells. Cells were transiently transfected with Nck-GFP, Lck and the indicated HA-tagged TSAd constructs. Cell lysates and anti-Nck IP were separated by SDS-PAGE and immunoblotted with indicated antibodies. Data are representative of three independent experiments