Wbox2 binds to both AP2 and SNX9. (A) ITC measurements of peptide binding to SNX9 and β2 hinge + appendage domain (HAD). Heat curves recorded as a function of time during successive 1.9-µl injection of peptides into the cell containing buffer, SNX9, or β2-HAD. Left: Wbox2 + buffer (1.8 mM Wbox2 injected into reaction buffer); TAT + SNX9 (1.8 mM TAT injected into 50 µM SNX9); Wbox2_mutant + SNX9 (1.8 mM Wbox2_mutant injected into 50 µM SNX9); Wbox2 + SNX9 (1.8 mM Wbox2 injected into 50 µM SNX9). Right: Wbox2_mutant + β2-HAD (1.2 mM Wbox2_mutant injected into 200 µM β2-HAD); Wbox2 + β2-HAD (1.2 mM Wbox2 injected into 200 µM β2-HAD). Heat curves were executed with NITPIC and fitted with SEDPHAT. The fitting model for Wbox2 + SNX9 is A+B+B <–> AB+B <–> BAB, with two nonsymmetric sitesK. The fitting model for Wbox2 + β2-HAD is A+B <–> AB, hetero-association. (B and C) GST-TD (1–580) pulldown of AP2 from mouse brain extract in the presence of increasing concentrations of TAT, Wbox2_mutant, and Wbox2. Representative Western blot images are presented in B and quantified in C. GST-TD was used as loading control, and each data point represents the average and SD from three independent runs.