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Fig. 1. The µstpCys motif prevents secretion of TPO and targets it to ERAD. (A) Schematic presentation of the Myc-tagged TPO vectors. (B) COS-7 or HEK293T cells transfected with vectors encoding Myc-tagged TPO-µstpCys (Cys) or TPO-µstpSer (Ser) were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time. Cells and medium were separated, cells were lysed and substrates were immunoprecipitated (IP) from lysed cells or medium by an antibody against Myc. Immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([³⁵S]). (C) The graph, representative of five independent experiments, illustrates the amounts of TPO-µstpCys (Cys; open symbols) or TPO-µstpSer (Ser; filled symbols) remaining in HEK293T cells (circles), recovered from medium (squares) or their sum (triangles). Amounts estimated by densitometry of autoradiograms in B were calculated as a percentage of their levels at the end of the pulse (100%). (D) HEK293T cells transfected with a vector encoding Myc-tagged TPO-µstpCys were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine, chased for the indicated time with (+) or without (–) ALLN and MG-132 and analyzed as described in B. (E) The graph, representative of five independent experiments, illustrates the amounts of TPO-µstpCys remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) HEK293T cells. The amounts detected in cells (circles), recovered from medium (squares) or their sum (triangles) were estimated by densitometry of autoradiogram D, calculated as a percentage of their levels at the end of the pulse (100%), and the half-life values (see text) were calculated.

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