Fig. 6: Segregation enables paracellular ion flux over the TJ meshwork. | Nature Communications

Fig. 6: Segregation enables paracellular ion flux over the TJ meshwork.

From: Nanoscale segregation of channel and barrier claudins enables paracellular ion flux

Fig. 6

a Representative confocal images of MDCKII QKO cells stably expressing (sT) FLAG-Cldn2, FLAG-Cldn10a, and FLAG-Cldn2+FLAG-Cldn10a. Single claudin expressing cells were immunostained with anti-Cldn2 (magenta; 2nd-Atto647N) or anti-Cldn10 (magenta; 2nd-Atto647N) and anti-ZO-1 (yellow; 2nd-AF594) antibody. Double claudin expressing cells were immunostained with anti-Cldn2 (magenta; 2nd-Atto647N) and anti-Cldn10 (yellow; 2nd-AF594). The white rectangle indicates the region of interest for the magnification in (b). b Magnification of FLAG-Cldn2+FLAG-Cldn10a expressing cells from (a). White arrows point out differently sized TJ parts that contain only Cldn10a. c Representative confocal images of MDCKII QKO sT FLAG-Cldn3, FLAG-Cldn15 and FLAG-Cldn3+FLAG-Cldn15. Single and double claudin expressing cells were stained with anti-Cldn3 (magenta; 2nd-Atto647N) or anti-Cldn15 (magenta; 2nd-Atto647N) and anti-ZO-1 (yellow; 2nd-AF594) antibody. d Summary of the TER (ohm*cm2), fluorescein flux (10−6 cm/s) and PNa/PCl ratio from the measured cell lines: MDCKII, MDCKII QKO and MDCKII QKO sT FLAG-tagged Cldn2, Cldn3, Cldn10a, Cldn15 and MDCKII QKO sT FLAG-tagged Cldn2 + Cldn10a and FLAG-tagged Cldn3 + Cldn15. Data represent the mean ± SD from 3 to 5 independent experiments. Single data points are shown in Supplementary Fig. 12. e Absolute permeability for sodium (PNa; magenta bars) and chloride (PCl; cyan bars) of MDCKII, MDCKII QKO and MDCKII QKO sT FLAG-tagged Cldn2, Cldn3, Cldn10a, Cldn15, and MDCKII QKO sT FLAG-tagged Cldn2 + Cldn10a and FLAG-tagged Cldn3 + Cldn15. Data represent the mean ± SD. For the electrophysiological measurements every n represents one transwell filter; n(MDCKII) = 14; n(MDCKII QKO) = 14; n(FLAG-Cldn2) = 9; n(FLAG-Cldn10a) = 11; n(FLAG-Cldn2+FLAG-Cldn10a) = 8; n(FLAG-Cldn3) = 9; n(FLAG-Cldn15) = 12; n(FLAG-Cldn3+FLAG-Cldn15) = 9; from 3–5 independent experiments; one-way ANOVA with Kruskal–Wallis test; ***P ≤ 0.001, **P ≤ 0.01, ns (not significant). The total claudin expression level was determined by immunoblotting for Cldn2, Cldn3, Cldn10, and Cldn15 (20–25 kDa). β-actin (40 kDa) served as control. All representative images derive from 3 independent experiments. Scale bars, 10 μm (a, c), 2 μm (b). Source data are provided as a Source data file.

Back to article page