Fig. 4

LRP5 promotes TGF-β/Smad signaling in renal tubule epithelial cells. a, b Western blot analyses of p-Smad2/3 and total Smad2/3 (a) in WT PTECs and Lrp5^−/− PTECs treated with 2 ng/ml TGF-β1 for the indicated times, and (b) in HKC-8 cells after transfection of a control plasmid (pcDNA3) or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for the indicated times. c Immunostaining of Smad2/3 (red color; scale bar = 25 μm) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min. d Western blot analyses and (e) densitometry quantification of nuclear levels of p-Smad2/3 and Smad2/3 in the fractions extracted from HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 60 min (n = 6). f 3TP-Lux luciferase activity assay in HKC-8 cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 hr, followed by the treatment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of pcDNA3 control (n = 3). g, h Western blot analyses of fibronectin, CTGF, and α-SMA (g) in primary PTECs from WT and Lrp5^−/− mice exposed to the indicated concentrations of TGF-β1 for 24 h, and (h) in HKC-8 cells after transfection of a pcDNA3 plasmid or a plasmid expressing LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 24 h. Representative images are shown from at least 3 independent experiments. All values are expressed as mean ± SEM. **p < 0.01, by two-way ANOVA with pair-wise multiple comparisons