Figure 4.

kAE1 protein is in close proximity and interacts with claudin-4. (A) Confluent mIMCD3 cells stably expressing kAE1 protein in an inducible manner were grown for 10 days in a 10 cm dish, lysed (Input) and claudin-4 was immunoprecipitated using a rabbit anti-claudin-4 antibody (IP lanes). As a control an irrelevant IgG antibody was used (right panel). Eluted proteins were separated on SDS-PAGE gel prior to immunoblotting with a mouse anti-claudin-4 (bottom blot) or mouse anti-HA (top blot) antibody. Dark circle corresponds to kAE1 carrying complex oligosaccharide, white circle indicates kAE1 carrying high mannose oligosaccharide. (B) A similar experiment was performed but immunoprecipitating claudin-3. No kAE1 co-immunoprecipitated with claudin-3 (top panel). (C) The claudin-4 immunoprecipitation was repeated using mIMCD3 kAE1 E681Q cells and shows that claudin-4 interacts with the mutant. (D) Proximity ligation assay was performed on mIMCD3 cells stably expressing kAE1 grown for 10 days on semi-permeable filters. CNX, corresponding to calnexin, was used as a positive control¹⁵, Cldn-4 corresponds to claudin-4. Cells were examined under a confocal microscope using a 63 X objective and sections (X-Z) or top views (X-Y) are shown. Red signal indicates that the 2 proteins labeled are within 30 to 40 nm of distance from each other. Nuclear staining is shown in blue as DAPI staining.