A CRISPR screen reveals the genetic requirement for the ER _GFP _NS degradation pathway. (A) A Venn diagram shows the number of genes whose inactivation increased ER_GFP_NS and ER_GFP_K20, respectively. LFC, log fold change. (B) A STRING network analysis reveals gene networks that affect the steady-state levels for both ER_GFP_NS and ER_GFP_K20. (C) STRING networks affecting only the level of ER_GFP_NS. Node color in B and C indicates fold enrichment in GFP-high populations. (D) HEK293T cells were transfected with control, Hrd1, or VCP/p97 siRNA together with ER_GFP_NS. Cells were imaged by fluorescence confocal microscopy to measure the ER_GFP_NS levels (left) or by immunoblotting (right) to confirm gene knockdown. Scale bar, 5 μm. (E) Quantification of two independent experiments as shown in D. Error bars, SEM, ****P < 0.0001 by unpaired Student’s t test. (F) HEK293T cells were transfected with the indicated siRNAs followed by ER_GFP_NS. Cells were stained by Alexa⁵⁹⁴-labeled cholera toxin subunit B to identify the Golgi. Scale bar, 5 μm. (G) Quantification of the percentage of transfected cells showing a Golgi localization of ER_GFP_NS (left) or the total fluorescence intensity of ER_GFP_NS (right) from three independent experiments. Error bars, SEM. *P < 0.05; **P < 0.01; ****P < 0.0001 by one-way ANOVA’s Dunnett’s multiple comparison test. Source data are available for this figure: SourceData F3.