BLTP2^Halo shows heterogeneous localization in different cell types. (A–C) Localization of BLTP2^Halo in U2OS cells (A), COS-7 cells (B), and MDA-MB-231 cells (C). BLTP2^Halo co-localizes throughout the ER with ER marker Sec61β in all these cells. In U2OS and MDA-MB-231 cells, it shows an additional enrichment at ER-PM contacts. (D) Small puncta of anti-V5 immunoreactivity are present both in unedited HeLaM cells and in HeLaM-KI^2XV5, showing that such staining is nonspecific. (E) In some MDA-MB-231 cells, BLTP2^Halo shows an enrichment on tubular structures similar to those observed in HeLaM cells. (F) BLTP2^Halo mainly localizes throughout the ER in COS-7, and only occasionally it is enriched at some tubular structures. (G) BLTP2-positive tubules in HeLaM cells are positive for Rab8A. (H) A fluorescently labeled MHC-I antibody added to HeLaM cells expressing BLTP^EGFP labels the tubules.