Multimodal Analysis of Cellular Signaling and Oxidative Stress

Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF) and dihydroethidium (DHE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed as described elsewhere^{20 (link),23 (link),30 (link),49 (link)}. The antibodies used for IHC and IF included the following: anti-Hhip (monoclonal clone 5D11), α-smooth muscle actin (α-SMA) and β-actin antibodies from Sigma-Aldrich Canada; Shh, TGFβ receptor II (TGFβRII), CD31, synaptopodin (Synpo) (P-19) and p57 (H-91) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); p27^Kip1 antibody from BD Biosciences (San Jose, CA, USA); cleaved caspase-3 (Asp175) and caspase-3 antibodies from Cell Signaling (Danvers, MA, USA); anti-TGFβ1 antibody from R&D Systems, Inc. (Burlington, ON, Canada); Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) antibody (New England Biolabs, Whitby, ON, Canada); Smad2/3 antibody (Cedarlane-Millipore, Burlington, ON, Canada); and anti-NADPH oxidase 4 (Nox4) antibody (Abcam, Cambridge, MA, USA). GKT137831 (dual inhibitor of both Nox1 and Nox4) was procured from Cayman Chemical (Ann Arbor, MI, USA).