LDVICp016 is a secreted protein. The optimised DNA sequence encoding LDVICp016L was synthesised in vitro (GeneArt, Invitrogen) and subcloned into the pcDNA V5His (Invitrogen) expression plasmid. As a positive control, the secreted vTNFR from ectromelia virus CrmD cloned into the same plasmid backbone was used. Panel shows a western blot analysis using anti-V5 tag antibody (Invitrogen) of equivalent amounts of whole cell extracts or extracellular media corresponding to 293 T cells transfected with a control plasmid (pcDNA) in lane 1 or with the plasmid for expression of CrmD or LDVICp016L in lanes 2 and 3, respectively and harvested at 48 hours post-transfection. To control for lack of contamination of extracellular media with cell contents, samples were analysed with an anti-tubulin antibody. The position of molecular mass markers are shown.