Figure 2.

PP2A-B56 and Knl1 are dispensable for Bod1 recruitment to kinetochores. HeLa cells were treated with either control siRNA or a smart pool siRNA targeting all PP2A-B56 isoforms as described previously [11]. After 48 h cells were fixed in paraformaldehyde and stained with (a) the total Bod1 peptide antibody or (b) a PP2A-B56α isoform-specific antibody. Metaphase control cells or B56siRNA-treated cells showing the characteristic B56-depletion phenotype of metaphase chromosome alignment defects were imaged. The rightmost panels are magnifications of the same cell (region indicated by white boxes). Total (c) Bod1 and (d) B56α intensities at kinetochores were quantified. (e) Immunoblot corresponding to the B56-depletion experiments in (a–d) using antibodies against two of the five targeted B56 isoforms and vinculin as a loading control. (f) HeLa cells were treated with either control or Knl1 siRNA for 48 h, fixed in paraformaldehyde, and co-stained with both total Bod1 peptide antibody and a Knl1-specific antibody. Metaphase cells were imaged for both treatment conditions. The rightmost panels are magnifications of the same cell (section indicated by white boxes). Total (g) Bod1 and (h) Knl1 intensities at kinetochores were quantified. Single z-sections are shown for all images. Scale bars are 1 µm. Pairwise comparisons were evaluated by unpaired Student's t-test. Two-tailed p-values are shown. n = 10 cells per condition. Error bars represent standard error.