Ribosome UFMylation, SAYSD1, and the TRAPP complex facilitate ER _GFP _NS degradation. (A) Control or UFM1 knockout HEK293T cells were transfected with ER_GFP_NS and treated with the indicated inhibitors for 6 h. Cell extracts were analyzed by immunoblotting (IB). +CHO, glycosylated; −CHO, deglycosylated. (B) ER_GFP_NS-expressing cells treated as indicated were fractionated into a membrane (M) and cytosol (C) fraction before immunoblotting (left panel). Right panel, the membrane fraction used in lane 4 was treated with Endo H and then analyzed by immunoblotting. *, cleaved ER_GFP_NS fragments. (C) Top panels, HEK293T cells were transfected with the indicated siRNAs together with ER_GFP_NS. Cells were imaged 48 h after transfection. A fraction of the cells was analyzed by immunoblotting to confirm the knockdown efficiency (bottom right). The graph shows the quantification result from three independent experiments. N > 40 cells. ****P < 0.0001 by unpaired Student’s t test. (D) Control or UFM1 KO cells were transfected with either empty vector (EV) or ER_GFP_NS. Cells were lysed in CHAPS buffer. The lysates were subject to immunoprecipitation with GFP antibodies before immunoblotting. Note that the detergent in input samples affects the mobility of low molecular weight proteins. (E) As in D, except that RPL26ΔC and an isogenic WT line were used. (F) HEK293T cells transfected with the indicated siRNAs and ER_GFP_NS were analyzed by confocal microscopy. The numbers indicate the knockdown efficiency determined by qRT-PCR averaged from three independent experiments. (G) Quantification of experiments represented in F. At least 30 randomly selected cells from two independent experiments were analyzed. Error bars, SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA’s Dunnett’s multiple comparison test. IP, immunoprecipitated. Scale bars in C and F, 5 μm. Source data are available for this figure: SourceData F2.