GlyT2 expression after a pulse-chased in culture cells.

<p>COS7 cells transfected with GlyT2 cDNA in pCDNA3 were pulse-labeled for 15 min with [³⁵S]methionine/cysteine and chased for the times indicated in the conditions given in Material and Methods. The cells were then surface biotinylated, lysed and the protein lysate was either immunoprecipitated with GlyT2 antibody (total transporter, A) or bound to streptavidin-agarose and sequentially immunoprecipitated with GlyT2 antibody (biotinylated fraction, B). Proteins extracted from the beads were resolved in SDS-PAGE. (A) Kinetics of GlyT2 expression. (B) Kinetics of GlyT2 plasma membrane expression in the same cells as in A. Lower panels: (A) densitometry of the 100 kDa and 75 kDa bands in the fluorograms. (B) Biotinylated bands are represented as a percentage of each of the lanes labeled in A. Bars represent the S.E.M. (n = 3). (C) Cells were treated overnight with the vehicle alone (DMSO) or with 10 µg/ml tunicamycin, and then pulse-chased as described above. (D, E) Immunoprecipitates were treated overnight with the vehicle alone (endoglycosidase buffer, -) or with the indicated endoglycosidase (+) in denaturing conditions, and then resolved by SDS-PAGE as described in the Materials and Methods. The transporter proteins (100 kDa, 75 kDa and 60 kDa) are indicated with arrowheads.