Rabbit anti dkk1

MSCs were lysed on ice in lysis buffer containing 50 mmol·L⁻¹ Tris-HCl, 150 mmol·L⁻¹ NaCl, 1 mmol·L⁻¹ EDTA, 1% Nonidet P-40, and a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The cells were then centrifuged at 18 000×g for 15 min at 4 °C to remove the cell debris. The supernatants were heated at 95 °C for 5 min in sample buffer containing 2% SDS and 1% 2-mercaptoethanol, separated on 10% SDS–polyacrylamide gels, and transferred to PVDF membranes using a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% milk for 1 h and then incubated with rabbit anti-AFF1 (Bethyl, Montgomery, TX, USA, 1:1 000), rabbit anti-AFF4 (Abcam, Cambridge, MA, USA, 1:1 000), rabbit anti-DKK1 (Cell Signaling, Danvers, MA, USA, 1:1 000), rabbit anti-ID1 (Abcam, 1:1 000), or mouse anti-α-tubulin (Sigma, 1:5 000) overnight, followed by a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). The antibody–antigen complexes were visualized with SuperSignal reagents (Pierce, Rockford, IL, USA).

The following primary antibodies were used: mouse-anti-Cbfβ (Santa Cruz Biotechnology Cat# sc-56751, RRID:AB_781871), mouse-anti-Col2α1 (Santa Cruz Biotechnology Cat# sc-52658, RRID:AB_2082344), rabbit-anti-MMP13 (Abcam Cat# ab39012, RRID:AB_776416), rabbit-anti-ADAMTS5 (Santa Cruz Biotechnology Cat# sc-83186, RRID:AB_2242253), rabbit-anti-Sox9 (Santa Cruz Biotechnology Cat# sc-20095, RRID:AB_661282), rabbit-anti-Yap (Santa Cruz Biotechnology Cat# sc-15407, RRID:AB_2273277), rabbit-anti-Dkk1 (Cell Signaling Technology Cat# 48367, RRID:AB_2799337), and mouse-anti-Active-β-catenin(Millipore Cat# 05–665, RRID:AB_309887). Imaging was done with a Leica DMLB Microscope and a Leica D3000 fluorescent microscope and were quantified by Image J software.