BLTP2-positive tubular endosomes are positive for PM markers, and its C- terminal region is required for its PI4P-dependent tethering function. (A) The tubular structures of HeLaM cells are also positive for another PM marker, Lyn11-RFP. (B) BLTP2-positive tubules are also labeled by CellBrite at 4°C. (C) Sequence alignment of the C-terminal region of human BLTP2 with the corresponding region of other species. Conserved residues are highlighted in blue, with darker color indicating higher conservation. aa 2219–2300 of Drosophila BLTP2 (hobbit) are framed by red solid lines, and aa 2177–2235 of human BLTP2 by magenta dashed lines. (D and E) U2OS cells (D) and HeLaM cells (E) co-expressing the BLTP2 C-terminal deletion mutant BLTP2^EGFP(△59) and BLTP2^Halo FL. BLTP2^EGFP(△59) shows reduced localization relative to BLTP2^Halo FL at ER-PM contact sites in U2OS cells (arrowhead) and at tubular structures in HeLaM cells. (F) BLTP2 re-establishes contact with tubular endosomes after A1 washout.