NEMF1 facilitates ER _GFP _NS degradation via CAT tailing. (A) U2OS cells transfected with the indicated siRNA together with ER_GFP_NS were lysed and analyzed by immunoblotting to verify knockdown efficiency (indicated by the numbers). (B) U2OS cells transfected with the indicated siRNAs and ER_GFP_NS were stained with Hoechst and imaged. Scale bar, 10 µm. (C) Quantification of two independent experiments as shown in B. Error bars, SEM, ****P = 0.0001 by one-way ANOVA’s Dunnett’s multiple comparison test. (D) HEK293T cells transfected with the indicated siRNAs and ER_GFP_NS were treated with anisomycin (ANS, 200 nM) for 1 h. Cell lysates were analyzed by immunoblotting. (E) Quantification of ER_GFP_NS levels in three independent experiments represented by D. Error bars, SEM. (F) Quantification of the HMW smear level in the indicated knockdown cells. Error bars, SEM, **P < 0.01 by one-way ANOVA’s Dunnett’s multiple comparison test. (G) Schematic of the ER_GFP-_TEV–NS construct. (H) ER_GFP-_TEV–NS was immunoprecipitated (IP) from control or UFM1 knockdown HEK293T cells and treated with TEV protease (+) (0.01 µg/μl, 3 h) or mock treated (−) before immunoblotting (IB). Top panels show long (LE) and short (SE) exposures from two independent experiments. Lower panels show IB analyses of a fraction of the corresponding lysates. (I) Representative images of U2OS cells transfected with mCherry-tagged ER_TEV (mCh-ER_TEV) together with ER_GFP-_TEV–NS or ER_GFP-NS. Scale bar, 10 µm. (J) Quantification of GFP fluorescence in individual cells as shown in I. P values were by unpaired Student’s t test, n = 2 independent experiments. ns, not significant. (K) A model of distinct TAQC branches. Source data are available for this figure: SourceData F4.