Cytoplasmic events preceding HShh-C proteolysis. (A) To test for deglycosylation of HShh-C, HShh-HA was stably expressed in 293T cells. A cycloheximide chase was performed for 2 h in the presence or absence of the proteasome inhibitor MG132. Cell lysates were incubated in the absence or presence of protein N-glycanase F (PNGase F) as indicated. Samples were analyzed by SDS-PAGE and immunoblotting with HA antibodies. Immunoblotting with p97 antibodies served as a loading control. (B) To test for polyubiquitination of HShh, cells stably expressing HShh-HA were incubated in the absence or presence of MG132 for 2 h. Extracts were subjected to immunoprecipitation (IP) with HA antibodies, and the proteins were analyzed by SDS-PAGE and immunoblotting (IB) with HA-antibodies (lanes 3 and 4) or ubiquitin (Ub) antibodies (lanes 1 and 2). Lanes 5 and 6 show blots of the extract before immunoprecipitation. (C) To test whether Hrd1 polyubiquitinates HShh, an experiment as in B was performed except that, where indicated, cells were transfected with a Myc-tagged dominant-negative Hrd1 mutant (Myc-Hrd1-291A). To test for the presence of the Hrd1 mutant, the samples were also analyzed by blotting with Myc antibodies. Immunoblotting for p97 served as a loading control. (D) Cells stably expressing HShh-HA were transfected with Myc-tagged wild-type Hrd1 (Hrd1-WT) or catalytically inactive Hrd1 mutants (Hrd1 C291A or Hrd1 C291A-C307A). As a control, a Myc-tagged version of reticulon 4A was used. Cell extracts were either analyzed directly (lanes 1–4) or subjected to immunoprecipitation with HA antibodies (lanes 5–8). All samples were analyzed by SDS-PAGE and immunoblotting with HA or Myc antibodies. Molecular masses are given in kilodaltons.