(A) stx3 localizes strictly to the apical plasma membrane in WT cells. (B) stx3 abberantly localizes to a subapical compartment in TM9SF4-KO cells. (C) ANO8-KO cell lines display apical localization of stx3. (D) stx3 mislocalizes to large, basolateral, actin-rich compartments in PIP5K1C-KO cells, reminiscent of microvillus inclusions. (E) stx3 localizes to subapical compartments in ARHGAP33-KO cells. (F) stx3 mislocalizes to large, basolateral actin-rich compartments in MTMR2-KO cells. (G, H) GALNT2- and TMEM110-KO cell lines display apical localization of stx3. XY = top view of polarized monolayer; XZ/YZ = lateral view of polarized monolayer. (I) Delocalized stx3 in polarized CaCo2 cells was quantified (dot box plot, Mann–Whitney U test. **p<0.01, ***p<0.005, n.s., not significant, n ≥ 100 cells per condition). XY = top view of polarized monolayer; XZ/YZ = lateral view of polarized monolayer. Scale = 5 µm.