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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 1. — A pool of REEP4 localizes to the INM. (A and B) HeLa cells with genomically HA-tagged REEP4 immunolabeled for HA. (A) Costained for Reticulon 4. (B) Costained for the ER marker Calnexin and the INM marker Lamin B1. Nuclear rim signal is detected for all three proteins after Triton X-100 treatment, but only for Calnexin after digitonin permeabilization. (C) HEK293T cells expressing the ONM marker GFP-Nesprin1α were fractionated into nuclei and cytoplasm. Left: Nuclei treated with agarose-coupled or unmodified (free) proteinase K and analyzed by immunoblotting. Right: In the cytoplasmic fraction, agarose-proteinase K degraded REEP4 but not the ER luminal protein GRP94, suggesting that ER integrity is maintained. (D) TEV cleavage assay: Purified NusA-TEV cleaves only cytoplasmic HA tags, while nucleoplasmic HA tags are protected. V5 tags mark cells expressing the reporter. (E and F) NusA-TEV cleavage assays were performed on HeLa cells expressing REEP4-V5-tev-HA (E) or REEP3-V5-tev-HA (F). Cells were immunostained for the V5 tag, the HA tag, and Lamin A/C to identify the nuclear rim. In A, B, E, and F, scale bars are 10 µm. In E and F, asterisks indicate untransfected cells.