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. 2018 Jan 8;32(5):2615–2629. doi: 10.1096/fj.201700699R

Figure 7.

Figure 7.

Claudin-5 incorporation into TJs is rescued by Cx43 GJ inhibition. A) FRAP analysis of Claudin-5 (Cldn5) in control, CCM3KD, and CCM3KD + GAP27 (100 μm) cells expressing Claudin-5–AcGFP. B) Super-resolution imaging of TJ-associated Claudin-5 (arrows). C) Immunofluorescence staining of Claudin-5 and ZO-1 in control, CCM3KD, and CCM3KD + GAP27 (100 μM) cells. Scale bar, 25 μm. D) Quantitation of the average TJ-associated Claudin-5 fragment length in Claudin-5/ZO-1 costained immunofluorescent images. Fragment length was measured in ImageJ as described for ZO-1 fragment length in Fig. 5C. *P < 0.05, ***P < 0.0001 compared with control, ***P < 0.0001 compared with CCM3KD. E) FRET analysis of TJ-associated Claudin-5 and ZO-1 in cells coexpressing Claudin-5–mCherry and ZO-1–AcGFP. The number of cell-border ROIs giving a FRET signal (FRET+ ROI) was measured as a percent of the total number of cell-border ROIs analyzed [n = 20 total ROIs (4 ROIs/cell), 2 independent experiments]. F) FRET analysis of Claudin-5 transinteraction. Cells were separately transfected with either Claudin-5–AcGFP or Claudin-5–mCherry at 1:1 concentration, then seeded together. ROIs in which neighboring cells exhibited incorporation of Claudin-5–AcGFP and Claudin-5–mCherry into a shared TJ were selected for FRET analysis. FRET was performed and analyzed as described in Fig. 6E for 15 ROIs (3 ROIs/cell) and 2 independent experiments.