FIGURE 2.

Mint2 interacts with TrkA in yeast two-hybrid and GST pulldown assays. A, the interaction of Mint2 with TrkA^IC was analyzed by filter assay for LacZ reporter gene β-galactosidase activity and leucine-deficient growth assay for LEU reporter gene expression. The indicated Mint2 domains fused in-frame into pB42AD vector were cotransformed with TrkA^IC (pGilda-TrkA^IC), a kinase-dead TrkA^IC (pGilda-TrkA^IC (K547N)), the intracellular domain of the epidermal growth factor receptor (pGilda-EGFR^IC), or the vector alone (pGilda) into the yeast reporter strain EGY48. Positive and negative controls were described in methods. B, PC12 cells expressing the indicated constructs, either treated with NGF or left untreated, were lysed and subjected to GST pulldown followed by Western blotting (IB) with anti-TrkA antibody (Chemicon). The whole cell lysate is shown as an input control. C, various GST fusion proteins were incubated with PC12 cell extracts containing endogenous TrkA. After GST pulldown, GST complexes were immunoblotted with anti-TrkA (Santa Cruz) for Western blot analysis. 1, GST-Mint2-N (68 kDa); 2, GST-PTB (45 kDa); 3, GST-PDZ1 (34 kDa); 4, GST-PDZ2 (34 kDa); 5, GST (negative control; 27 kDa); 6, PC12 cell lysate (positive control).