Figure 1. Gremlin exists both as a monomer and a covalent dimer.

A. total lysates from healthy murine organs were immunoprecipitated with anti-gremlin antibody, separated by SDS-PAGE under non-reducing conditions and probed with anti-gremlin antibody. Black arrow, gremlin dimer; open arrow, gremlin monomer. IgG were used as a control. B. FGF2-T-MAE cells were treated with increasing concentrations of H₂O₂ for 1 hour. At the end of incubation, the cells were incubated for 4 hours with fresh medium. Conditioned medium was collected and immunoprecipitated with anti-gremlin antibody. Immunoprecipitated fractions were analysed by WB under non-reducing conditions. C-D. recombinant his-tagged gremlin^WT was transiently expressed in HEK293T cells, purified by IMAC and analyzed by SDS-PAGE followed by Western blotting (WB) under non-reducing (C) or by analytical size exclusion chromatography. The elution profile of gremlin was obtained by dot blot analysis of the eluted fractions (D). Black arrows indicate the retention volume of standard proteins (seroalbumin: 132 and 66 kDa; ovalbumin: 45 kDa; carbonic anhydrase: 29 kDa and lactoalbumin: 14.2 kDa). E. IMAC purified gremlin was further subjected to heparin-affinity chromatography. Heparin column was washed with a discontinuous NaCl gradient. Eluted fractions were separated by SDS-PAGE under non-reducing conditions and probed with anti-gremlin antibody. Black arrow, gremlin dimer; open arrow, gremlin monomer. F. pure dimeric gremlin^WT was analysed by SDS-PAGE followed by Western blotting (WB) under non-reducing (−βME) and reducing (+βME) conditions and by silver staining (SS) of the gel.