Fig. 2

Role of C-terminal domain of hAQP5 in protein trafficking. (A) Schematic representations of SNAP-hAQP5 constructs (full length wildtype (1-265)), created with BioRender.com. (B) WB analysis for SNAP tag (using anti-SNAP tag antibodies) of NS-SV-AC cells stably transfected with SNAP-hAQP5 (1-265), (1-245), (1-241) and (1-227). STD: molecular weight standards. (C-D-E-F) Violin plots (with median and interquartile range of 25–75% percentile) of the membrane upper quartile (UpQ) intensity. Cells were pretreated for 24 h with 50µM indomethacin prior to treatment for 8 h without (CTRL) or with 10 µM FK, 0.1 µM TH, or both (FK + TH), in 3 independent experiments. Statistical significance evaluated using Kruskal-Wallis test with post-hoc Dunn’s tests is indicated as follows: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. Schematic representations of SNAP-hAQP5 constructs were created with BioRender.com. Representative confocal images of immunofluorescent staining with anti-SNAP antibodies (red) and DAPI (blue). (C) In SNAP-hAQP5 (1-245), the medians with IQR are: 1 with IQR 0.724–1.375, n = 389 cells for CTRL; FK: 0.866 with IQR 0.648–1.158, n = 483 cells; TH: 0.644 with IQR 0.171–0.975, n = 410 cells and 1.257 with IQR 0.904–1.724, n = 428 for FK + TH. (D) In SNAP-hAQP5(1-241), the medians with IQR are: 1 with IQR 0.749–1.447, n = 431 cells for CTRL; FK: 1.058 with IQR 0.764–1.566, n = 535 cells; TH: 0.794 with IQR 0.530–1.160, n = 307 cells and FK + TH: 1.290 with IQR 0.927–1.798, n = 349 cells. (E) In SNAP-hAQP5 (1-227), the medians with IQR are: 1 with IQR 0.750–1.448, n = 513 cells for CTRL; FK: 0.974 with IQR 0.713–1.327, n = 518 cells, TH: 0.995 with IQR 0.699–1.534, n = 503 cells and FK + TH: 0.902 with IQR 0.617–1.296, n = 597 cells. The specificity of the anti-SNAP antibodies is shown in Fig. S6A