Fig. 5. The µstpCys N-glycan decelerates degradation of TPO. HEK293T were transfected with an empty vector (mock), or with vectors encoding Myc-tagged TPO-µstpCys (WT), TPO-µstpCysKR (KR) or TPO-µstpCysNQ (NQ). In the NQ mutant, the only N residue in the µstp motif was replaced by Q, abolishing the µstp glycosylation site. (A) Cells were lysed, samples of cell lysate (10%) were collected and substrates were immunoprecipitated (IP) from the remaining 90% of the lysate by an antibody against Myc. Immunoprecipitates were treated with (+) or without (–) endo H, and immunoprecipitates and cell lysates were resolved by SDS-PAGE and immunoblotted (IB) with an antibody against µstp. Fully glycosylated WT and KR, under-glycosylated NQ and endo-H-treated de-glycosylated substrates are indicated. (B) HEK293T transfected with an empty vector (mock) or with vectors encoding Myc-tagged TPO-µstpCys (WT) or TPO-µstpCysNQ (NQ) were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine, chased for the indicated time, lysed and substrates were immunoprecipitated (IP) by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([³⁵S]; upper panels) to monitor degradation and then immunoblotted (IB) with an antibody against µstp (lower panels) to estimate steady-state levels. (C) The graph, representative of three independent experiments, illustrates the amounts of TPO-µstpCys (WT; circles) or TPO-µstpCysNQ (NQ; triangles) remaining in HEK293T cells. The amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of their levels at the end of the pulse (100%), and half-life values (see text) were calculated.