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. 2013 Dec;183(6):1945–1959. doi: 10.1016/j.ajpath.2013.08.026

Figure 5.

Figure 5

Def-6 expression is enhanced in PKCλ/ι−/− glomeruli and localizes to cellular edges in podocytes. A–C: Staining of kidney cryosections from PKCλ/ι+/+ and PKCλ/ι−/− mice. A: Upper panels: Overview staining with an antibody against Def-6 (×10 magnification) reveals strong glomerular expression. Lower panels: Control staining with secondary antibody or Def-6 blocking peptide validated the specificity of the Def-6 antibody. Scale bars = 200 μm. B: Co-staining of Def-6 (red) and the podocyte marker podocalyxin (green) reveals enhanced podocytic expression of Def-6 in PKCλ/ι−/− glomeruli (boxed areas in Merge views, and arrowheads in Detail views). Nuclei are visualized with DAPI (blue). Scale bars = 25 μm (applies to all panels). C: For quantification of Def-6 expression, sections were double-stained against Def-6 (red) and nidogen (green). Nuclei were visualized using DAPI (blue). Def-6 expression is enhanced in the podocytic areas of PKCλ/ι−/− glomeruli (boxed areas in Merge views, and arrowheads in Detail views). Scale bars = 25 μm (applies to all panels). D: Images of PKCλ/ι+/+ and PKCλ/ι−/− glomeruli (40 glomeruli from four animals of each genotype) were obtained and analyzed using a semiquantitative score ranging from 0 (no expression) to 4 (strong expression). Compared with WT glomeruli, PKCλ/ι−/− glomeruli show a significantly higher score and an obvious change in score distribution. n = 4 mice of each genotype, ≥10 glomeruli per mouse. ∗∗∗P < 0.001 E: Endogenous Def-6 localizes to the cellular edges and co-localizes with vinculin, in particular in the PKCλ/ι−/− podocytes. Immunostaining of differentiated podocytes with an antibody against Def-6 (red) and vinculin (green) (insets in Merge views, and arrows in Detail views). Scale bar = 50 μm (applies to all panels). F: Def-6 membrane localization is significantly enhanced in PKCλ/ι−/− podocytes. Subcellular fractionation of differentiated PKCλ/ι+/+ and PKCλ/ι−/− podocytes. Lysates were normalized with Giα3 (membrane fraction, M), Sp1 (nuclear fraction, N), and GAPDH (cytosolic fraction, C). Quantitative analysis of Def-6 expression in three independent experiments of two independent cell clones. ∗∗P < 0.01. G: Isolation of plasma membranes from PKCλ/ι+/+ and PKCλ/ι−/− podocytes reveals increased localization of Def-6 in plasma membranes of PKCλ/ι−/− podocytes in comparison with WT cells on Western blot analysis (left panel). For quantification, lysates of three independent experiments from two independent cell clones were normalized with Giα3 (right panel). ∗P < 0.05.