Fig. 2

THTR-1 protein is N-glycosylated on asparagine 63. (A) NIH3T3 cells were transfected as described in Figure 1 legend, except that after transfection, cells were incubated at 28°C for 40 hr. Cell lysates were prepared and proteins were resolved on a 10% SDS-PAGE and THTR-1 expression was followed as described in Figure 1. Where indicated, cells were treated with tunicamycin (10 µg/ml) for 24 hr prior to cell harvesting. [S³⁵]-methionine-labeled THTR-1 G172D in vitro translated protein product (G172D IVT) represents the migration of the core THTR-1 protein with no posttranslational modifications (lane 5). (B) Cell lysates (50 µg protein) derived from THTR-1-transfected cells either with the wild-type (THTR) or mutant G172D THTR-1 expression plasmids, as indicated, was incubated for 45 min at 37°C in the absence (+buffer) or presence of 2000 units of PNGase F (+PNGase F). Following PNGase F treatment, SDS-sample buffer was added and the extract was subjected to 10% SDS-PAGE followed by Western blotting. In vitro translated product (G172D IVT) represents the migration of the core unglycosylated THTR-1 protein (lane 7). (C). NIH3T3 cells were transfected with different expression plasmids encoding for wild-type THTR-1 and the indicated mutants. Transfected cells were grown at either 37°C (lanes 1–3) or 28°C (lanes 4–6) and proteins from in vitro THTR-1 translated product (lane 7) were separated by SDS-PAGE followed by Western blotting with anti-Myc 9E11 monoclonal antibody. (D) Swiss 3T3 cells were transfected with different expression plasmids encoding for wild-type THTR-1 and the indicated mutants grown at 28°C. Cell lysate and Western blotting were performed as described in (A).