Figure 3.

Components of the ERAD Tuning Pathway Are Associated with DMVs

(A) Cell extracts from Atg7^+/+ (lane 1) and Atg7^−/− (lane 2) were separated by SDS-PAGE and western blot membranes probed with antibodies against EDEM1, p62, LC3, and tubulin. Repetition of the analysis showed no significant differences in the EDEM1 level in Atg7^+/+ versus Atg7^−/− MEF. The percentages (right) indicate the relative EDEM1 levels in the knockout cells compared to wild-type cells and represent the average of two experiments.

(B) Atg7^+/+ and Atg7^−/− MEF were metabolically labeled and chased for the times indicated before lysis and EDEM1 immuno-isolation. The residual radiolabeled EDEM1 present in each lane was quantified and indicated below each band. Repetition of the analysis showed no significant differences in the EDEM1 turnover in Atg7^+/+ versus Atg7^−/− MEF.

(C) Atg7^+/+ MEF were untreated or treated with 100 mM CQ or 1 mM rapamycin (rap) for 4 hr before preparation of cell extracts and analysis as in (A). The percentages (right) indicate the relative EDEM1 levels in drug-treated cells compared to mock-treated cells and represent the average of two experiments.

(D) Same as (B) to confirm, in a pulse-chase radiolabeling experiment, that CQ and rap delay EDEM1 turnover.

(E and F) HeLa cells were infected with MHV-Srec and processed for IF at 7 hr p.i. using antibodies against dsRNA and (E) EDEM1 or (F) OS-9.

(G) Atg7^+/+ and Atg7^−/− MEF infected with MHV-Srec were fixed at 7 hr p.i. and processed for IF using antibodies against OS-9 and dsRNA.

(H) Cell extracts were analyzed by western blot using antibodies against EDEM1 or OS-9. The percentages (right) indicate the relative EDEM1 and OS-9 levels in infected cells compared to control cells and represent the average of two experiments.

See also Figures S2, S3, and S4.