Figure 5.

LCK SH3 and ITK SH3 may bind simultaneously to TSAD aa 242–268. A–C, LCK SH3 interacts with both TSAD aa 239–256 and aa 257–274. Additional mutation of Pro²⁴⁷ or Pro²⁶³ further reduces binding. A and B, pulldown experiment in 293T cells using the same TSAD constructs as in Fig. 2, E and G, respectively. Pulled down proteins and lysates were probed with the indicated antibodies. C, the graph represents ImageJ analysis of data shown in A and B (n = 3, mean ± S.D. (error bars)). D, sequences of custom synthesized TSAD peptides aa 242–268, 242–256, and 254–268. E, in vitro pulldown experiment using a 10 μm concentration of each SH3 domain and increasing amounts of TSAD aa 242–268 (10, 20, and 40 μm) in a total volume of 100 μl. The GST-ITK SH3 domain was added while attached to glutathione Sepharose^TM beads. To eliminate bead loss, a 4-fold amount of GST-glutathione Sepharose^TM beads was added to the mixture, as is evidenced from the bottom panel. F, in vitro pulldown experiment performed as in D, using a 10 μm concentration of each SH3 domain, and a 10 μm concentration of the indicated TSAD peptides. WB, Western blot.