Immunohistochemistry and immunoblotting were performed as previously described [8 (link)]. Protein concentrations were determined by the BCA Protein Assay Kit (Beyotime, Shanghai, China). Primary antibodies used for immunohistochemistry and/or immunoblotting were as follow: α-SMA, Collagen I, Collagen III, CCN3, TGF-β, p-RAF, p-MEK, p-ERK, E-cadherin, Vimentin and Actin (Abcam, Cambridge, MA, USA). Recombinant human CCN3 was purchased from Peprotech (Rocky Hill, NJ, USA). TGF-β from LX2 was quantified by ELISA (R&D Labs, Minneapolis, MN, USA). Assays were performed according to the manufacturer’s instructions in quadruplicate as previously described [9 (link)].
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