Modulation of TGF-β1 and Neuropeptide Signaling

TGF-β1 (R&D Systems, code: 240-B-002), reconstituted in 4mM hydrochloric acid and 0.1% bovine serum albumin (BSA, Sigma, code: A9647) was used at a concentration of 1 ng/ml based on modification from Le Roux et al’s study involving human keratocytes [11 (link)]. The TGFβRI/II kinase inhibitor LY2109761 (Santa Cruz, code: sc-396262) was used at a concentration of 2 μmol/l based on Xu et al’s study [18 (link)]. LY2109761, prepared in dimethyl sulphoxide (DMSO), was used to block the effects of TGF-β1, and was added 20 min prior to TGF-β1 incubation. In addition, SP (Calbiochem, code: 05-23-0600) at 10⁻⁷ M and NK-1 R receptor blocker (L-733.060) (Tocris, code: 1145) at 10⁻⁶ M were used in concentrations as previously used in Backman et al’s study involving tenocytes [8 (link)]. SP at 10⁻⁷ M has also been used in studies involving human colonocytes [19 (link)] and human lung epithelial cell lines [10 (link)]. The concentrations of the mAChR blocker atropine at 10⁻⁵ M (Sigma, code: A0132) and ACh at 10⁻⁶ M (Sigma, code: A661) were based on our previous study involving human tenocytes [7 (link)].