Constitutively internalized LGR5 internalizes into VPS26-positive endosomes and is deposited to the TGN.
A–C, HEK 293T cells were transiently transfected with a 3×HA N-terminally epitope-tagged WT-LGR5. Cells were pulsed with an HA antibody for 45 min on ice, washed, chased for 0, 5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with appropriate primary and secondary antibodies to visualize HA (A–C, red), VPS26 (A, green), M6PR (B, green), or Trip230 (C, green). D–F, HEK cells were transfected with a 3×HA N-terminally epitope-tagged human V2R, pulsed with an αHA antibody for 45 min on ice, washed, chased for 0, 5, 15, 30, or 120 min at 37 °C in the absence (−) or presence (+) of arginine vasopressin (0.1 IU/ml), fixed, permeabilized, and stained with appropriate primary and secondary antibodies to visualize HA (D–F, red), M6PR (D and E, green), or Vps26 (F, green). Merged 100× confocal images are presented.