The macrophage cDNA libraries in the prey vector pPR3-N used for yeast two-hybrid screening were constructed according to a split-ubiquitin system (Dualsystems Biotech). The Vsig4 encoding sequence were obtained by PCR with specific primers with Sfi I restriction site at respective 5′ terminals and cloned into bait vector pBT3-SUC. The bait constructs were transformed into the yeast reporter strain NMY51 via standard procedures. For screening, the library plasmids were introduced into the yeast cells harboring bait constructs. After 3 days, transformants were grown on selective medium lacking leucine, tryptophan, histidine, and adenine, with addition of 20 mM 3-AT. Library plasmids were isolated from positive clones and retransformed into NMY51 to test bait dependency. Only preys that activated the histidine and adenine reporters in the presence of VSIG4 and not pBT3-SUC were considered true interactors.

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