Fig. 4

Transforming growth factor beta type I receptor (TβRI)/endothelin-1 receptor A (ETAR) co-immunoprecipitation. a TβRI was immunoprecipitated (IP) and its association with ETAR was assessed by Western blot (WB). The immunoprecipitation assay showed an association between ETAR and TβRI, independent of both transforming growth factor beta (TGF-β) and endothelin-1 (ET-1) stimulation. In systemic sclerosis (SSc) fibroblasts (FBs), the levels of co-immunoprecipitated receptors were significantly higher when compared with healthy control (HC) FBs. Blot was representative of all the experiments. Co-immunoprecipitated protein bands were quantified by densitometry and the values are expressed as protein relative quantification/TβRI relative quantification. b Western blot of ETAR protein. In SSc FBs, ETAR expression was significantly higher when compared with HC FBs. Blot was representative of all the experiments. The protein bands were quantified by densitometry and the values are expressed as protein relative quantification/β actin relative quantification. c SSc FBs were transfected with specific ETAR siRNA or nontargeting scramble scr-siRNA, and ETAR expression was evaluated by quantitative RT-PCR. The cells transfected with ETAR siRNA showed a decreased expression of ETAR gene, when compared with cells transfected with scr-siRNA. d Western blot of phospho-Sma and Mad Related (pSMAD)1/5 and pSMAD2/3. In SSc FBs treated with scr-siRNA, TGF-β induced a significant increase in SMAD phosphorylation, and macitentan (MAC) inhibited this effect. In SSc FBs treated with ETAR siRNA, TGF-β induced a significant increase in SMAD phosphorylation, and MAC failed to inhibit this effect. Pictures are representative of all experiments. Protein bands were quantified by densitometry and the values were expressed as protein relative quantification/β actin relative quantification. **p = 0.0002, ***p = 0.0001. UT untreated