(A–C) Characterization of an antibody to the first extracellular loop of Cldn16 (α-loop) and endogenous Cldn16 expression in MDCK cells. MDCK cells were incubated for 1 hour at 37°C in the presence of anti-loop antibody alone (A), together with antigenic peptide (B), or with preimmune serum (C). Cells were then washed, fixed, and permeabilized, and the anti-loop antibody was detected by immunofluorescence microscopy using a labeled secondary antibody. (D–F) Detection of HA-tagged Cldn16 expressed in MDCK cells. MDCK cells expressing N-terminally HA epitope–tagged Cldn16 were incubated for 1 hour at 37°C with anti-loop antibody. α-HA (D) and the anti-loop antibody (E) were then detected in permeabilized cells. (F) Merging the HA and anti-loop antibody staining shows extensive colocalization at the cell surface and in a few intracellular vesicles. (G–I) Detection of anti-loop antibody internalized via HA-tagged Cldn16 in early endosomes of transfected HeLa cells. Anti-loop antibody (G) and EEA1 (H) were visualized by immunofluorescence microscopy, and the 2 images were merged (I). (J–L) Clathrin-mediated internalization of Cldn16. HeLa cells expressing HA-tagged Cldn16 were incubated for 1 hour at 37°C with anti-loop antibody under either hypertonic (J) or cytosol acidified (K) conditions to disrupt clathrin function or in the presence of cholesterol oxidase (L) to disrupt caveolae. Cldn16 was then visualized by immunofluorescence microscopy. In L, cells were stained with antibodies to EEA1, and the merged image is shown. Shown are representative images of 2–3 independent experiments.