Af1096

Secreted DKK1 levels were measured either by Western blot after immunoprecipitating DKK1 from the culture medium using anti-DKK1 (#AF1096; R&D Systems) or by commercially available ELISA assay (Human Dkk-1 Quantikine ELISA Kit, #DKK100B; R&D Systems) according to the manufacturer’s protocol. For DKK1 ELISA, absorbance was measured at 450 nm with a correction at 540 nm using the Eon microplate spectrophotometer (Agilent).

SDS–PAGE gels were blotted onto nitrocellulose membranes and incubated with the respective primary antibodies (anti-SFRP1, Cell Signaling Technologies, clone D5A7, 1:500; anti-DKK1, R&D Systems, AF1096, 1:2,000). As secondary antibodies fluorescently labelled IRDye800 and 680 (LI-COR, Lincoln, NE) were used. Fluorescence detection was performed using an Odyssey infrared imaging system (LI-COR) and blots were further analysed using the Odyssey v3.0 software (LI-COR).

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).