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. 2020 Dec;190(12):2436–2452. doi: 10.1016/j.ajpath.2020.08.015

Figure 1.

Figure 1

Expression of mutant uromodulin (Umod) in kidney epithelial cell cultures demonstrates increased basolateral secretion of aggregated protein. A: Western blot analysis of Umod using LLC-PK1 cell lysates with stable transfection of wild-type (WT) and/or mutant (mut) uromodulin expression plasmids as indicated. Control (cntrl) cells were transfected with empty plasmid. B: Western blot analysis of uromodulin using 10× concentrated cell culture medium removed from apical (A) and basolateral (BL) sides of stable transfected LLC-PK1 cells as indicated. C: Western blot analysis of uromodulin using 10× concentrated cell culture medium removed from apical chamber and untreated (−) or treated (+) with PNGase. Untreated medium from cells transfected with WT and mutant plasmids was added for size comparison. D: Confocal images of uromodulin immunofluorescence using LLC-PK1 cells transfected as indicated. Left column: Apical membrane plane. Right column: Lateral plane. E: Nondenaturing Western blot analysis of uromodulin using A and BL medium (arrow indicates high-molecular-weight protein; and arrowheads indicate uromodulin multimers) and corresponding electron microscopy images. Scale bars: 5 μm (D); 100 nm (E). sup, supernatant.