Fig. 3. EDEM accelerates the degradation of misfolded A1AT glycoprotein. (A) Autoradiogram following SDS–PAGE of immunoprecipitated material from HEK 293 cells cotransfected with NHK and EDEM-HA, or transfected with NHK and mock (indicated by –). The position of the predicted Man9 form of the A1AT variant (NHK) is shown by *, and that of the Man8 form by **. Cells were pulse-labelled for 15 min with [³⁵S]methionine, and chased for the periods indicated. In this experiment only, lysates of cells cotransfected with EDEM-HA containing 1.6-fold more TCA-insoluble radioactivity than mock-transfected cells were used, since if equal amounts were used, the intensity of NHK signals in cells cotransfected with EDEM-HA was reduced to 42 ± 5% of the signal in mock-transfected cells at 0 h chase [see (C)]. The relative radioactivity in NHK at different times of chase was plotted on a semi-log scale relative to the intensity observed at 0 h chase. Error bars indicate standard deviations from the average of four (mock) or six (EDEM-HA) independent experiments. (B) Autoradiogram showing coimmunoprecipitation of NHK and EDEM-HA. The gel was exposed heavily to show coimmunoprecipitated bands. An arrowhead indicates the position of NHK, arrows show the transfected EDEM-HA, and the open triangle denotes the 150 kDa associated protein. The non-specific band detected in the immunoprecipitates using protein G-Sepharose is shown by dots (··). EDEM-HA appears as a doublet of different glycosylated forms, since a single band was formed after PNGase F treatment (data not shown), and EDEM has five possible N-glycosylation sites (Figure 1A). Molecular mass standards (kDa) are shown on the left. (C) Inhibition of NHK degradation by proteasome inhibitors. Cells were pulse-labelled for 15 min (P) and chased for 90 min (C), in the presence or absence of the proteasome inhibitors, lactacystin or MG132. MG135, a calpain inhibitor, was used as a control of MG132 treatment (indicated by –). Signals from immunoprecipitated NHK were compared with those of mock-transfected cells without proteasome inhibitor during pulse-labelling. (D) Autoradiogram showing coimmunoprecipitation of NHK and EDEM-HA in cells treated with lactacystin. An arrowhead, arrows, open triangle and dots indicate the same species as in Figure 3B. Open circle denotes another coimmunoprecipitated bands. The right panel (lanes 9–16) shows a 2.5 times longer exposure than the gel shown in the left panel (lanes 1–8).