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Fig. 2. The SH3 domain of cortactin is sufficient to activate N-WASP. (A) Western blot (left) of GST pull-down assays showing association of GST-cortactin fusions with endogenous N-WASP. TCL, total cell lysate; 1/40th of the amount of lysate used in the pull down was used for TCL. Right panel shows comparative size of GST fusion proteins used in this study on Coomassie-stained gels. Values down the side of the gel show the positions of protein standards in kDa. (B-E) Pyrene Assays. 10 nM Arp2/3 complex, 1.5 µM pyrene-labeled G-actin and 25 nM GST-N-WASP or His₆-N-WASP were incubated together with GST-tagged or untagged cortactin proteins at the specified concentrations. (B) Optimal activation of GST-N-WASP by GST-cortactin proteins (GST, 1.3 µM; GST-Ct, 1.3 µM; GST-SH3, 1 µM). (C) Fold activation of GST-N-WASP by GST, GST-Ct or GST-SH3 cortactin, or GST-Nck. All fold activation curves are a logarithmic `best fit' of the data points shown. Inset shows N-WASP binding to GST-Ct, GST-SH3 and GST-Nck beads. (D) GST tag on N-WASP is not required for N-WASP activation by cortactin. Sample polymerization curves are shown for each GST-cortactin protein at the concentration giving optimal N-WASP activation in the pyrene assay (GST, 250 nM; GST-Ct, 1 µM; GST-SH3, 250 nM). (E) GST tag is not required for N-WASP activation by cortactin C-terminus. Plots show fold activation of GST-N-WASP by GST, GST-Ct, untagged cortactin C-terminus (Ct) or buffer control (Buffer).

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