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. 2010 Mar;176(3):1139–1147. doi: 10.2353/ajpath.2010.090459

Figure 6.

Figure 6

BMP-7 prevents TGF-β-mediated down-regulation of the repressor of Smad3 transcription, SnoN. A–C: HK-2 cells were incubated with 1 ng/ml TGF-β, 50 ng/ml BMP-7, or both, for time points to 24 hours before immunoblotting of whole-cell extracts. Subsequently, blots were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to confirm approximately equal loading. A: SnoN and Ski expression after TGF-β. B: SnoN expression after BMP-7. C: SnoN expression after TGF-β and BMP-7. D and E: HK-2 cells were incubated with 50 ng/ml BMP-7 or control medium for 24 hours before incubation with 1 ng/ml TGF-β or control medium for 30 minutes and immunoblotting of whole-cell extracts for SnoN. Subsequently, blots were reprobed for GAPDH to confirm approximately equal loading. D: Representative immunoblot of four experiments giving similar results. E: Densitometry of four independent experiments. F: HK-2 cells were transfected with SnoN siRNA together with Smad3 responsive (Scr) (CAGA luciferase) and control (Renilla) vectors for 48 hours. Subsequently, cells were incubated with BMP-7 or control medium for 24 hours and then incubated with TGF-β for 6 hours. Data are presented as firefly luciferase/Renilla luciferase, normalized to the TGF-β control. G: HK-2 cells were transfected with Ski siRNA together with Smad3-responsive (Scr) (CAGA luciferase) and control (Renilla) vectors for 48 hours. Subsequently, cells were incubated with BMP-7 or control medium for 24 hours and then incubated with TGF-β for 6 hours. Data are presented as firefly luciferase/Renilla luciferase, normalized to TGF-β control. RLU, relative light units.