Fig. 2. Production of recombinant Eda-A1 proteins. (A) Schematic drawing of the Eda-A1 protein with a transmembrane domain (TM), a furin cleavage site (asterisk), collagen like Gly-X-Y repeats (COL) and the globular TNF homology domain (TNF-DOMAIN). Figures refer to amino acid numbering of endogenous Eda-A1. Recombinant Eda-A1 starts at amino acid residue 180 and contains an N-terminal 6x His tag. Control protein contained Y343C mutation that abolishes receptor binding. Recombinant Fc-Eda-A1 consists of an N-terminal HA tag, an Fc-domain and the TNF-domain of Eda-A1. (B) Cos cells were transfected with the soluble, truncated form of wild-type Rec Eda-A1 (lane 2) or mutated Y343C Eda-A1 construct (lane 3), or were mock transfected (lane 1). Cell supernatants were analysed by western blot using an anti-Eda polyclonal antibody. (C) Purified Fc-Eda-A1 (5 µg) was analysed by SDS-PAGE under reducing conditions and stained with Coomassie Blue. The doublet band results from glycosylation. Molecular weight markers are indicated on the left.