Fig. 2. The µstpCys element prevents secretion of ssYFP and targets it to ERAD. (A) Schematic presentation of the Myc-tagged ssYFP vectors. (B) Hela cells expressing a vector encoding Myc-tagged ssYFP (myc), or HEK293 cell vectors encoding ssYFP-µstpCys (Cys) or ssYFP-µstpSer (Ser), were incubated with (+) or without (–) ALLN and MG-132, pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. Cells and medium were separated, cells were lysed and substrates were immunoprecipitated (IP) from lysed cells or medium by antibodies against GFP or Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([³⁵S]). ssYFP-µstpCys without N-glycan (^*), resulting from de-glycosylation or inefficient glycosylation. ssYFP-µstpSer with high-mannose (arrow) or complex (arrowhead) N-glycans are indicated. (C) The graphs, representative of three independent experiments, illustrate the amounts of the indicated ssYFP fusion proteins remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) cells. The amounts detected in cells (circles) or recovered from medium (squares) were estimated by densitometry of autoradiograms in B, calculated as a percentage of their levels at the end of the pulse (100%), and the half-life values (see text) were calculated.