Following internalization SSTR2 traffics to a syntaxin-6–positive compartment through an endosomal pathway. (A–C) AtT20 cells were serum starved for 2 h and then transferred to ice. The cells were incubated on ice for 1 h in the presence of 5 µg/ml Alexa Fluor 647–labeled Trf and 100 nM [D-Trp⁸]-SOM. Cells were than fixed (0 min) or transferred to 37°C for either 5 or 40 min as indicated before being fixed and processed for immunofluorescence with antibodies recognizing SSTR2 and syntaxin-6. Images were acquired using a LSM 710 confocal microscope. The images in B and C correspond to the boxed areas in A. For C, the intensity distributions for the lines passing through the images are indicated. Scale bars represent 10 µm in A and 5 µm in B and C. (D) AtT20 cells were washed extensively with Earle’s buffer and then incubated with 80 µM dynasore or vehicle control for 20 min before and during a 40-min incubation without (no stimulation) or with 100 nM [D-Trp8]-SOM at 37°C. Cells were than fixed and processed for immunofluorescence with antibodies recognizing SSTR2 and syntaxin-6. (E) Sucrose (0.45 M) was added to cells in Earle’s buffer and incubated for 15 min before and during a 40-min stimulation without (no stimulation) or with 100 nM of [D-Trp⁸]-SOM at 37°C. The stimulation was ended by washing in ice-cold Earle’s buffer and the cells were processed for immunofluorescence with antibodies recognizing SSTR2 and syntaxin-6. Images were acquired using the superresolution mode of the LSM 880 confocal microscope. Scale bars represent 5 µm.