Aurora B phosphorylates YY1 at serine 184 in vivo.

<p>(<b>A</b>) HEK293 cells were synchronized in mitosis with nocodazole block for 17 hours. After the block, the cells were treated with the Aurora inhibitor VX-680, with the indicated concentrations for 15 minutes. Cell lysates were prepared, followed by Western blot. The blot was probed with anti-pS184 antibody with relative levels indicated below, then stripped and reprobed with anti-YY1 antibody followed by anti-cyclin B1antibody to show proper mitotic synchrony. (<b>B</b>) HEK293 cells were plated, cultured overnight, and then transfected with 20 nm control scrambled siRNA or Aurora B siRNA. After 48 hours of knockdown, the cells were lysed, and extracts were analyzed by Western blotting. The blot was probed with anti-pS184 antibody then stripped and reprobed with anti-YY1 antibody followed by anti-Aurora B antibody. (<b>C</b>) Co-immunoprecipitation of YY1 with Aurora A and Aurora B from HEK293 cells transiently transfected with HA-Aurora A, Flag-Aurora B and no transfection (control) followed by nocodazole block. Aurora A was immunoprecipitated using anti-HA antibody and Aurora B was immunoprecipitated using anti-Flag mouse MAb cross-linked to resin beads. Non-transfected cells were also immunprecipitated using anti-HA antibody and anti-Flag mouse MAb cross-linked to resin beads, which were used as a control for the specificity of the immunoprecipitation. Western blot analysis was performed on the immunoprecipitated samples and probed with anti-YY1 antibody. Input samples were probed with anti-YY1 antibody, anti-HA antibody and anti-Flag antibody.