YPD is a potent trafficking domain that targets exogenous channels and truncated proteins to ZGs in AR42J cells. (A) Schematic diagrams of HA-tagged WT rat AQP4-M23 isoform (RnAQP4-M23-WT-HA) and mutant in which the two SMs in the C-terminal amino acid sequence, a YSM and diLeu SM, were first erased and the other substituted by the YPD of rat AQP12 (RnAQP4-M23-ΔSMs-YPD-HA). On the right is the diagram of HA-tagged truncated HsAQP12 bearing only the last transmembrane domain (TMD6) and the complete C terminus (HsAQP12-CT-HA). (B–E) Confocal fluorescent images of dexamethasone-differentiated AR42J cells transiently transfected with empty pcDNA3 vector (B), or expressing RnAQP4-M23-WT-HA (C), RnAQP4-M23-ΔSMs-YPD-HA (D), or HsAQP12-CT-HA (E), and incubated with CCK-8. The aquaporin channels (red) and AMY (green) are stained with the α-HA and a-AMY antibodies, respectively. The images show the compilation of Z-stack series into orthogonal projections (reconstructed 3D image). (F–H) Line-scan profiles (white bar in the merged images, left panels) of fluorescence intensity for the analysis of colocalization of the different constructs (red curves) and AMY (green curves). (I and J) Immunoblots of TE and purified ZG from AR42J cells transfected with the pcDNA3 empty plasmid or expressing RnAQP4-M23-WT-HA or mutant construct (I), or HsAQP12-CT-HA (J), as indicated. The different polypeptide constructs were detected using the α-HA, and protein loading was normalized with the α-AMY antibody. Molecular mass markers (kDa) are on the left. (K) Confocal fluorescent images of AR42J cells transiently transfected with RnAQP4-M23-WT-HA and exposed to CCK-8 as in B-E stained with α-HA antibody (red) and an antibody against the active form of caspase-3 (green). The images show the compilation of Z-stack series into orthogonal projections (reconstructed 3D image). WT, wild type; TE, total extracts; diLeu SM, dileucine SM. Source data are available for this figure: SourceData F9.