5-HT3A/BBS receptors are constitutively internalized in both HEK-293 and N1E-115 cells. a) HEK-293 and N1E-115 expressing 5-HT3A/BBS receptors incubated with BTX/555 (red label) for 30 min at 4 °C. b) After labeling surface 5-HT3A/BBS receptors, HEK-293 and N1E-115 cells were incubated at 37 °C for 30 min in the absence of an agonist, and subsequently imaged. Arrows indicate internal puncta in XY, XZ, and YZ images. c) Following the internalization of labeled receptors at 37 °C, HEK-293 and N1E-115 cells were incubated in the presence of BTX/488 (green label) for 30 min at 4 °C then fixed and imaged. Arrows indicate previously internalized BTX/555. d) To test the temperature sensitivity of the internalization, surface 5-HT3A/BBS receptors were labeled with BTX/555 (red) in HEK-293 and N1E-115 cells and were incubated at 4 °C for an additional 30 min in the absence of BTX. Following this incubation cells were labeled with BTX/488 (green). Scale bars=10 μm.