Figure 4.

T/P dimerizes in vitro and dimerization requires the HLH domain. cDNA constructs for the indicated mutations of T/P were cloned into the pcDNA3 expression vector, and in vitro transcription/translation was performed according to the manufacturer’s instruction (Promega TNT kit) using radiolabeled [³⁵S]methionine. Quantity of DNA added was adjusted to give approximately equal amounts of each translated protein. For lanes 7–12, one-half of the reaction mixture was removed and immunoprecipitated with an antisera to the carboxyl terminus of human PDGFβR. Total reaction products or total immunoprecipitates were separated by SDS/PAGE. The gel was fixed, treated with Enhance (Amersham), dried, and exposed to film at −70° for 30 min.