Rab10 is involved in SSTR2 recycling. (A) HeLa cells expressing EGFP-SSTR2 along with mCherry-Rab8, mCherry-Rab10, or mCherry-Rab13 were imaged live, with the figure revealing cropped frames from Videos 7–9. The boxed area from the Rab10-transfected cells is shown at higher magnification below. Arrows indicate SSTR2/Rab10-positive vesicles moving toward and fusing with the plasma membrane, which is indicated by the dashed line. Scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively. (B) Representative immunoblots from wild-type (WT) AtT20 cells and two Rab10-knockout (KO) cell lines generated using CRISPR/Cas9. The migration of molecular weight markers in kilodaltons is indicated. (C) AtT20 cells, either wild-type, two Rab10 knockout lines (KO-1 and KO-2), and an isogenic control were left untreated (no stimulation) or were treated for 60 min with [D-Trp⁸]-SOM to induce maximal SSTR2 internalization, followed by a brief acid wash and incubation in ligand-free media for 1 h at 37°C, without or with 100 nM CRF. Following the treatments, cells were fixed and processed for immunofluorescence with antibody recognizing SSTR2. Scale bar represents 5 µm. (D) Quantification of SSTR2 cell surface immunofluorescence intensity of three successive experiments as in C. Data were analyzed by two-way ANOVA and Bonferroni comparison test (procedures implemented in GraphPad statistical package). Data are presented as least-square means ± SEMs with treatment effects significant at 0.05. **, P ≤ 0.01; ***, P ≤ 0.001.