Fig. 7. A reduction of erasin levels inhibits ERAD. (A) HEK293 cell cultures were cotransfected with constant amounts of HA-CD3, together with either ERASIN cDNA expression plasmid, or empty vector plasmid, or with 10 nm SMARTpool ERASIN siRNAs. For the knockdown experiments, the cells were transfected with the siRNAs 20 hours before transfection with CD3. 20 hours after CD3 transfection, cycloheximide was added to all cultures and protein lysates were collected at the time intervals indicated. Equal amounts of protein lysates were then immunoblotted for erasin, HA and actin. The arrow and the asterisk indicate the glycosylated and non-glycosylated forms of CD3, respectively. (B) Densitometric analysis of the bands shown in A and expressed relative to the level present at the 0 time point. The Microsoft Excel program was used to produce a best-fit line for each experimental set. (C) A HEK293 cell line stably expressing GFP^u was transfected with the nucleic acids as described in A, but omitting HA-CD3. The turnover of GFP^u in the different transfected cells was determined by classical pulse-chase studies of [³⁵S]methionine-labeled and immunoprecipitated GFP proteins over a 7-hour period (upper panel). Immunoblot analysis of the cell lysates used for the immunoprecipitation studies confirmed that erasin levels were altered in the expected manner (middle panel), with actin loading of these lysates confirming equal protein loading of the lysates (bottom panel). (D) Graph showing the exponential decline of pulse-labeled GFP^u protein over time. The turnover of GFP^u was altered little in the three experimental sets.