Partial segregation of M1– and M23–AQP4 in lamellipodial and somatic regions of cells expressing both isoforms. (A) Schematic showing predicted distribution of M1–AQP4–GFP and M23–AQP4–mCherry in migrating cells and TIRF images of U87-MG cells transfected with both isoforms then fixed and stained with phalloidin showing localization of M1– and M23–AQP4 in relation to an actin enriched membrane extension. (B) Ratio of M1/M23 fluorescence intensity in somatic and lamellipodial regions of individual U87-MG cells. **, P < 0.01 by paired t test, n = 8. (C) Schematic showing predicted distribution of labeling with monoclonal antibodies (mAbs) specific for only orthogonal arrays (r93) or for all AQP4 (r58) in migrating cells expressing both M1– and M23–AQP4 and confocal images demonstrating that mAb r93 does not bind to cells transfected with only M1–AQP4, whereas r58 binds to cells transfected with M1– or M23–AQP4. (D) TIRF images showing labeling of F-actin, total AQP4, and r93 or r58 mAbs in migrating U87-MG cells transfected with M1– and M23–AQP4. (E) Quantification of the relative amount of AQP4 stained with each mAb in lamellipodial compared with somatic regions of cells stained with r58 (n = 5) or r93 (n = 6). ***, P < 0.001 between r58 and r93 groups by t test.