Binding of Ang-1.3-kb isoform protein to 1.5-kb isoform protein in vitro.
(A) The GST–Ang-1.5-kb fusion protein was incubated with35S-labeled 1.3 kb, 0.9 kb, or luciferase prepared by transcription/translation in vitro. Proteins bound to the 1.5-kb fusion protein were identified by incubation with glutathione Sepharose 4B beads, elution, electrophoresis on 12% SDS-PAGE, and autoradiography of the gel. Lane 1 (1.3 kb), lane 2 (0.9 kb), lane 3 (control luciferase protein). (B) cos cells were transfected with either a mixture of 1.5 and 1.3 kb containing vectors or 1.5-kb vector alone. Cells were lysed in lysis buffer, and isoforms were precipitated with or without antibody reactive with the 1.5-kb isoform, not the 1.3-kb isoform, plus protein G beads. Samples were then analyzed by Western blot with an antibody reactive with both the 1.5- and the 1.3-kb isoforms. Lane 1, 1.5- and 1.3-kb transfectant without antibody but with protein G beads; lane 2, 1.5-kb transfectant without antibody but with beads; lane 3, 1.5- and 1.3-kb transfectant with antibody plus beads; lane 4, 1.5-kb transfectant with antibody plus beads.
