Screening and identification of candidate receptors for AT. HEK293T cells were cotransfected with plasmid vectors expressing a tethered AT ligand and respective candidate receptor. The tethered AT ligand consists of whole AT sequence, linker, transmembrane region, and 3 hemagglutinin (HA) tag intracellularly. To each candidate receptor molecule was added the 3 Flag tag intracellularly. Detailed construct information was described in “Materials and methods.” After 24 hours of the transfection, the cell pellets were prepared and lysed by M-PER reagent. The lysates were incubated with agarose beads conjugated with monoclonal anti-HA Ab to pull down the expressed AT. (A) Summary of screening for candidate receptors for AT using the coexpression system. (B) The resulting immunoprecipitates were applied to SDS-PAGE. Western blotting using monoclonal anti-Flag Ab was then performed after transferring proteins onto PVDF membrane to detect AT-bound receptor candidate. The results obtained were repeated twice. Positive bands including CLEC1A are indicated by asterisks (∗∗∗, strong positive; ∗∗, weak positive; ∗, very weak positive). PVDF, polyvinylidene fluoride; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.