Figure 1.

A) hBVR binds and stimulates Akt1 kinase activity in cell. Cells were transfected with pcDNA-Akt1 with or without pcDNA-HA-hBVR or pcDNA3 vector. Some cells, as indicated, were treated with 40 ng/ml IGF-1. Immunoprecipitates were prepared using anti-Akt1 antibody, and Akt1 kinase activity was measured using Crosstide as substrate. Western blot of immunoprecipitated protein was probed with anti-Akt1. Expression of hBVR and Akt1 was verified by Western blot test. ***P < 0.001. B) hBVR and Akt1 coimmunoprecipitate. HEK cells overexpressing HA-hBVR either alone or cotransfected with pcDNA-Akt1 were treated with 40 ng/ml IGF-1 for 15 min; cell lysates were subjected to immunoprecipitation using anti-HA antibody. Immunoprecipitates were analyzed by Western blot analysis with anti-Akt1 antibody followed by anti-hBVR. Expression of hBVR and Akt1 was verified by Western blot test. C) hBVR and Akt association is mediated by Akt PH domain. GST-BVR bound to GSH agarose was added to lysates of cells that had been transfected with indicated EGFP fusion plasmids. Proteins bound by immobilized BVR were assayed by Western blot test, which was probed with anti-GFP antibodies. GST was included as additional control.