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. 2021 Dec 7;221(2):e202101049. doi: 10.1083/jcb.202101049

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© 2021 Golchoubian et al.

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Figure 2. — REEP4 BioID experiment identifies ELYS. (A) NusA-TEV cleavage assay on HeLa cells expressing REEP4-TurboID-V5-tev-HA reveals that REEP4-TurboID-V5-tev-HA localizes to peripheral ER and INM like endogenous REEP4. Asterisks indicate untransfected cells. (B) Scheme for REEP4-BioID with controls. (C) HEK cells were induced for expression of the indicated TurboID constructs for 24 h and treated with biotin for 1 h before fixation. TurboID constructs were detected with an HA antibody, and biotinylated proteins with fluorescently labeled streptavidin. (D) Proteins identified as proximal to REEP4 with fivefold or larger enrichment in REEP4 over control samples. See Table S1 for full list, raw MS datasets, and enrichment of proteins in REEP4 sample over single control samples. (E–G) REEP4-HA cells immunolabeled for the HA tag and ELYS and imaged by STED microscopy. (H) Quantification of REEP4 fraction in the vicinity of NPCs; ELYS included as a control. Scale bars are 10 µm (A and C), 5 µm (E), 500 nm (F), and 200 nm (G).