Figure 2.

Biochemical and biological analysis of T3/Y291F mutant. (A) T3/WT or T3/Y291F expression plasmids were transiently transfected into 293T cells. Cell extracts were immunoprecipitated (IP) with the antibodies anti-TRK, anti-Shc or with p13suc1-agarose and blotted with anti-phosphotyrosine, anti-TRK, anti-Shc and anti-FRS2 antibodies. (B) Effect of the Y291F mutation on TRK-T3 transforming activity. NIH3T3 cells were transfected with the indicated constructs and subjected to G418 and foci selection. Plates were fixed and scored after GIEMSA staining 2 weeks later. (C) Effect of the Y291F mutation on TRK-T3 differentiating activity. PC12 cells were transfected with the indicated constructs and scored for the presence of neurites 3 days later. As control, untreated and NGF-stimulated (50 ng ml⁻¹) PC12 cells are shown. The graph shows a quantification of differentiating activity. TRK-T3 constructs were cotransfected with VGF8-luc and Renilla plasmids; both luciferase activities were measured using the Dual-Luciferase reported assay system (Promega). The values were expressed relative to Renilla luciferase activity for normalization. The results presented are an average of three experiments.