Fig 1: Hirudin inhibits TGF-ß-induced PAR1 upregulation in HK-2 cells and the renal tissues of UUO mice. Relative (A) mRNA levels and (B) protein expression levels of S1PR1-4 and PAR1 in HK-2 cells were detected by RT-qPCR and western blot analysis, respectively. (C) Expression levels of proteins, including S1PR1-4 and PAR1, in the renal tissues of UUO mice were determined by western blotting. (D) Relative mRNA levels of PAR1 in HK-2 cells were determined by RT-qPCR. PAR1 expression in (E) HK-2 cells and (F) renal tissues of UUO mice was quantified by western blotting. Error bars represent the mean ± SEM from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. the control. #P<0.05, ##P<0.01 and ###P<0.001 vs. TGF-ß or UUO. UUO, unilateral ureteral ligation; PAR1, protease-activated receptor 1; S1PR, sphingosine-1-phosphate receptor; RT-q, reverse transcription-quantitative.
Fig 2: Hirudin suppresses TGF-β-induced epithelial-mesenchymal transition via S1P/S1PR2/S1PR3 signaling-mediated PAR1 inhibition. (A) Expression levels of N-cad, Slug and E-cad in HK-2 cells were determined using western blotting. (B and C) α-SMA expression in HK-2 cells was analyzed by immunofluorescence analysis. Scale bar, 100 µm. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ###P<0.001 vs. TGF-β. ∆P<0.05 and ∆∆∆P<0.001 vs. TGF-β+hirudun (1 mg/ml). S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1; N-cad, N-cadherin; E-cad, E-cadherin; α-SMA, α-smooth muscle actin; S1P, sphingosine-1-phosphate; TFL, TFLLR-NH2.
Fig 3: Hirudin suppresses TGF-ß-induced fibrosis and MCP-1 expression via S1P/S1PR2/S1PR3 signaling-mediated PAR1 inhibition. (A) Expression levels of collagen IV, FN and MMP-9 in HK-2 cells were determined by western blotting. (B) Relative mRNA level of MCP-1 in HK-2 cells was analyzed by reverse transcription-quantitative PCR. (C) MCP-1 expression in HK-2 cells was quantified by western blotting. Error bars represent the mean ± SEM from three independent experiments. ***P<0.001 vs. the control. ###P<0.001 vs. TGF-ß. ?P<0.05, ??P<0.01 and ???P<0.001 vs. TGF-ß+hirudun (1 mg/ml). MCP-1, monocyte chemoattractant protein 1; S1PR, sphingosine-1-phosphate receptor; PAR1, protease-activated receptor 1; FN, fibronectin; S1P, sphingosine-1-phosphate; TFL, TFLLR-NH2.
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