SGEF regulates AJ properties of epithelial cells. (A) Cell lysates from confluent CTRL and SGEF KD MDCK cells were analyzed by WB using anti-SGEF antibodies. Tubulin was used as a loading control. (B) Cell lysates from confluent CTRL, SGEF KD, and Rescue WT MDCK cells were probed with E-cadherin, Pan-cadherin, cadherin-6, β-catenin, p120-catenin, Scribble, and Dlg1 antibodies. Tubulin was used as a loading control. (C, C′, and D–F) IF showing the distribution of endogenous E-cadherin, p120-catenin, Scribble, Dlg1, β-catenin, and mNeon-SGEF (green) in CTRL, SGEF KD, and Rescue WT MDCK cells. The bottom panel in each set of images shows a zoomed image of the selected regions (dotted yellow line). Note that panels C and C′ show images from same field. Confocal images are maximum projections of apical Z-planes. Scale bars, top panels: 30 µm; bottom panels: 10 µm. (G) Linescan (6-µm line drawn perpendicular to center of junctions) of IF images in panels C to F. At least two fields from two independent experiments were used for quantification (≥200 junctions). The intensity profiles were manually centered around the highest peak for each condition. (H) XZ view of MDCK cells from CTRL, SGEF KD, and Rescue WT cells stained for E-cadherin (red), β-catenin (magenta), nucleus (blue) and mNeon-SGEF WT (green in merge panel). Scale bar, 10 µm. (I) Quantification of height in CTRL, SGEF KD, and Rescue MDCK cells. n = 50 cells for each condition from three independent experiments. Error bars represent min to max with all points. ****, P < 0.00005; ns, nonsignificant using Student’s t test (two-tailed, unpaired).