The SSTR2/syntaxin-6 compartment is resistant to Brefeldin A treatment. (A–C) AtT20 cells (A), cultures of primary cortical neurons (B), and C2C12 myoblasts (C) were incubated with vehicle control or Brefeldin A for 40 min before fixation and processing for immunofluorescence with antibodies recognizing the indicated antibodies. For B and C, the areas indicated by boxes in the merged images are shown at higher magnification on the right. Arrowheads represent nonoverlapping signals. For all images, scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively, and the dashed lines indicate cell boundaries. The outline of the nucleus (N) is also indicated by a dashed line in B. (D and E) AtT20 cells were incubated for 15 min with vehicle control or Brefeldin A before the 40-min stimulation of [D-Trp⁸]-SOM. The cells were then fixed and processed for immunofluorescence with antibodies recognizing the indicated proteins. The boxed areas are shown at higher magnification in the bottom sets of panels. The dashed lines indicate cell boundaries. Scale bars represent 5 µm and 1 µm for the low- and high-magnification images, respectively. (F) Pearson coefficient measurement of SSTR2/syntaxin-6 or SSTR2/PIST colocalization in a juxtanuclear ROI. The quantification is based on 40–60 cells per condition from four successive, independent experiments. Data were analyzed by one1-factor ANOVA and Tukey comparison test (procedures implemented in JMP statistical package; SAS Institute). Data are presented as least-square means ± SEMs with treatment effects significant at *, P < 0.05. (G–I) HEK-293 cells (G), HeLa cells (H), and MCF10A cells (I) were incubated with vehicle control or Brefeldin A for 40 min before fixation and processing for immunofluorescence using antibodies recognizing the indicated antibodies. Scale bars represent 5 µm. The dashed lines indicate cell boundaries.