Fig. 4. The µstpCys element, acting as a portable ERAD degron, does not serve as the ubiquitin conjugation site. HEK293T cells were transfected with vectors encoding (A) the wild-type (WT) version of the Myc-tagged TPO-µstpCys or (B) the TPO-µstpCysKR mutant (KR), in which the only K residue in µstp was replaced by arginine. Cells were pulse-labeled with [³⁵S]methionine-[³⁵S]cysteine and chased for the indicated time with (+) or without (–) ALLN and MG-132. Substrates were immunoprecipitated (IP) from lysed cells by an antibody against Myc, immunoprecipitates were resolved by SDS-PAGE, electroblotted and blots were exposed to autoradiography ([³⁵S]). (C) The graph, representative of three independent experiments, illustrates the amounts of TPO-µstpCys (WT; circles) or TPO-µstpCysKR (KR; triangles) remaining in untreated (open symbols) or ALLN- and MG-132-treated (+ Inhib; filled symbols) HEK293T cells. The amounts estimated by densitometry of autoradiograms A and B were calculated as a percentage of their levels at the end of the pulse (100%), and the half-life values (see text) were calculated.