Fig. 10.

Quality check of anti-pT232-Aurora-B antibody. (A) Dot-blot analysis with anti-pT232-Aurora-B polyclonal antibody. Phosphorylated and non-phosphorylated T232 peptides were blotted onto PVDF membranes at the indicated concentrations, followed by western blotting. (B) Peptide competition assay. After preincubation of anti-pT232 antibody with phosphorylated or non-phosphorylated T232 peptides, HeLa-S3 cells synchronized at metaphase were immunostained with preincubated antibody (red) and counterstained with anti-Aurora-B antibody (green). DIC, differential interference contrast. Scale bar, 10 μm. (C) HeLa-S3 cells were transfected with siRNAs against Aurora-B or firefly luciferase as a negative control (GL2). Cells synchronized at metaphase were co-immunostained with anti-pT232 (red) and anti–α-tubulin (green) antibodies. DNA was stained with Hoechst 33258 (blue). Arrowheads indicate the position of the metaphase plate. Scale bar, 10 μm.