Figure 7.
Expression of WT 3XFLAG-VRK1 results in a release of WT BAF from the insoluble nuclear matrix fraction of U2OS cells. (A) 35S-labeled 3XFLAG-BAF, synthesized using an vitro transcription/translation protocol, was incubated with DNA cellulose beads. Ten percent of the input (load, L) and all of the bound (B) protein were resolved electrophoretically and visualized by autoradiography. (B) U2OS cells transfected with a plasmid directing the expression of 3XFLAG-BAF were metabolically labeled with 32PPi from 20 to 24 h posttransfection. Cell lysates were subjected to immunoprecipitation with α-BAF antibody; immunoprecipitates were resolved electrophoretically and transferred to nitrocellulose. Total BAF protein was visualized by immunoblot analysis using α-BAF serum (left box); radiolabeled BAF was visualized by autoradiographic analysis of the same filter (right box). The open and filled arrowheads indicate the unphosphorylated and phosphorylated forms of BAF, respectively. (C) 3XFLAG-BAF, 3XFLAG-MAASQ, 3XFLAG-MTTAQ, and 3XFLAG-MAAAQ were expressed in U2OS cells by transient transfection and immunoprecipitated using rabbit α-FLAG antibody. Immunoprecipitates were then treated with λ-phosphatase (P'ase) or mock-treated and subjected to immunoblot analysis using mouse α-FLAG antibody. (D) U2OS cells were transfected with plasmids directing the expression of 3XFLAG-BAF or 3XFLAG-BAF-MAAAQ with or without plasmids directing the expression of hVRK1 or hVRK1-D177A. At 24 h posttransfection, cells were lysed in buffer containing 0.5% Triton X-100 and separated into soluble and insoluble fractions by low-speed centrifugation. Equal volumes of both fractions were subjected to immunoblot analysis with α-FLAG antibody. The open and filled arrowheads indicate the unphosphorylated and phosphorylated forms of BAF, respectively.