Figure 2.

Analysis and purification of expressed ORFV2-VEGF polypeptides. An ORFV2-VEGF-Flag was expressed in COS cells by transient transfection. (A) Cells were biosynthetically labeled with [³⁵S]Cys/Met for 4 h. Conditioned medium from these cells was immunoprecipitated with either M2-gel or control beads, and the washed beads were eluted with SDS/PAGE sample buffer under reducing (2% 2-mercaptoethanol) or nonreducing conditions. The single arrow indicates the nonreduced form of ORFV2-VEGF, and the double arrows indicate the two species of the reduced form. (B) Conditioned medium from COS cells transfected with ORFV2-VEGF DNA was purified on M2-gel and eluted with free Flag peptide. The fractions (1, void; 2, 3, and 4, purified protein) eluted from the M2-gel were collected, combined with 2× SDS/PAGE sample buffer (reducing), boiled, and resolved by using SDS/PAGE (4–20% gradient). Proteins were identified by using silver staining. Molecular weight markers are indicated. Purified proteins are indicated by arrows.