Protein expression and secretion
Splice variant cDNAs were subcloned into pcDNA3.1 (Invitrogen) with a Myc-His tag fused at the C-terminus of the proteins. To facilitate secretion, the native signal sequences of ASV derived from Met, FGFR1, VEGFR1, and RAGE were replaced by the tissue plasminogen activator signal/pro sequence (GenBank accession number NM_000930) by PCR cloning. All constructs were sequence verified, and were transiently expressed in HEK293 cells using LipofectAmine 2000 following the manufacturer's instruction (Invitrogen). Cell culture supernatants were collected 48 hours after transfection. To analyze expression of the recombinant proteins, equal amounts (20 μl) of supernatants were separated on SDS-PAGE gels. The separated proteins were transferred to nitrocellulose membranes, and were probed with anti-Myc antibody.