Fig. 1

THTR-1 expression in NIH3T3 cells transfected with the wild-type or the mutant G172D THTR-1 expression plasmids. (A) NIH 3T3 cells were transiently transfected with either a wild-type Myc-THTR-1 expression plasmid (THTR) or a mutant Myc-THTR-1 expression plasmid in which glycine 172 was substituted by an aspartic acid (THTR G172D). Cells transfected with an empty pCan expression vector (Vec.) were used as controls. Total cell lysates were prepared and proteins were resolved on a 10% SDS-PAGE followed by Western blotting using an anti-Myc 9E11 monoclonal antibody and anti-mouse IgG conjugated to HRP (Santa Cruz Inc., Santa Cruz, CA, USA) followed by chemiluminescence detection. Molecular weight (Mr) markers (ProSieve, BioWhittaker Molecular Applications Inc., Santa Cruz, CA, USA) are shown in kDa. (B) The indicated expression plasmids were used in a coupled in vitro transcription-translation kit using the T7 RNA polymerase in the presence of [³⁵S]-methionine. Proteins were then resolved on 10% SDS-PAGE followed by autoradiography.