SSTR2 displays slow recycling kinetics. (A) AtT20 cells were incubated for 40 min without (no stimulation) or with 100 nM [D-Trp⁸]-SOM and then fixed and processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6 (top two rows of panels). In parallel, cells treated with 100 nM [D-Trp⁸]-SOM for 40 min were subsequently transferred to 4°C, surface-bound agonist was stripped by a brief acid wash, and the cells were returned to 37°C for 1, 4, or 24 h before being processed for immunofluorescence with antibodies recognizing SSTR2 or syntaxin-6 (bottom three rows of panels). The boxes in the merged images are shown at higher magnification on the far right. Scale bars represent 5 µm and 1 µm for the low and high magnifications, respectively. (B) Quantification of SSTR2 cell surface immunofluorescence intensity from three successive experiments as shown in A and from three parallel experiments in the presence of Brefeldin A as shown in Fig. S1 with 10–15 cells quantified per condition per experiment. Data were analyzed by two-way ANOVA and Bonferroni comparison test (and procedures implemented in GraphPad statistical package). Data are presented as least-square means ± SEMs with treatment effects significant at 0.05. *, P ≤ 0.05; ***, P ≤ 0.001.