Western Blot Protein Analysis Protocol

Western blotting was performed as mentioned previously [46 (link)]. Briefly, protein samples were separated in 8–10% SDS-polyacrylamide gel, and then the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (IEVH85R; Millipore, Burlington, MA, USA). The primary antibodies against human or rat DKK1 (sc-25516; Santa Cruz, Dallas, TX, USA), mouse DKK1 (AF1096; R&D Systems, Minneapolis, MN, USA), fibronectin (ab45688; Abcam, Cambridge, UK), TGF-β1 (BS1361; Bioworld Technology, Dublin, OH, USA), α-smooth muscle actin (ab7817; Abcam, Cambridge, UK), β-catenin (#4970S; Cell Signaling, Boston, MA, USA), collagen IV (NB120-6586; Novus, Centennial, CO, US) and actin (#4970S; Cell Signaling, Boston, MA, USA) were used in this study. After removing the unbound primary antibody, the membranes were incubated with suitable secondary antibodies. The resultant antigen-antibody complexes were visualized using the Western Lighting Chemiluminescence Reagent (#NEL105001EA; PerkinElmer, Waltham, MA, USA).