Co-immunoprecipitation

PASMCs were stimulated with rhOPG (500 ng ml⁻¹) for 30 min at 37 °C. After stimulation, cells were lysed and the protein lysate concentration determined by a Pierce 660 nm protein assay. Co-immunoprecipitation was then performed using an anti-Fas or Ky3 antibody with human PASMC lysate and recombinant proteins, alongside negative controls, where antibodies were not added. ProteinG sepharose 4 Fast Flow beads (50% slurry) were added to each Co-IP reaction and immune complexes were precipitated. Each Co-IP reaction was then centrifuged and the pellet washed before re-suspending in sample reducing agent (NuPAGE, Life Sciences) with 5% v/v SDS and heating at 95°C. The supernatant was then analysed by western blotting. Membranes were incubated with goat polyclonal anti-OPG antibody (1:1000) (SC8468, Santa Cruz Biotechnology) or anti-Fas antibody (MA1–7622, Invitrogen) and IRDye 680LT Donkey anti-goat secondary antibody (1:15000) or IRDye 800CW donkey anti-mouse secondary antibody (1:15000) (Li-COR) to detect co-immunoprecipitated OPG. Membranes were scanned using the Li-COR Odyssey Sa system (LiCOR).