Characterization of Pds5 protein level and localization to chromosomes during meiosis. (A) Immunoblot analysis of Pds5 in wild-type (WT) and P_CLB2PDS5 cells during meiosis. Yeast cultures were induced to enter meiosis synchronously. Protein extracts were prepared at the indicated times. Pds5 was detected with a polyclonal anti-Pds5 antibody. β-Tubulin served as a loading control. (B) Meiotic nuclear division in wild-type and P_CLB2PDS5 cells. Cell aliquots were withdrawn at the indicated times, fixed with 4% paraformaldehyde, stained by DAPI, and visualized by fluorescence microscopy. Only minimal nuclear division was observed in P_CLB2PDS5 cells after 12 h of induction. (C) Pds5 and Rec8 colocalization to meiotic chromosomes is shown. Yeast nuclear spreads were prepared from synchronous meiotic cultures. Pds5 and Rec8-3HA were detected with anti-Pds5 and anti-HA antibodies (12CA5), respectively. Representative images from prophase I and anaphase I are shown. (D) ChIP assay of Pds5 and Rec8 binding to cohesin-associated regions at centromere 1 (CEN1), centromere 3 (CEN3), a cohesin site at the MAT locus (CARC3), and a cohesin site on chromosome XII (CARL2). Cells were arrested at pachytene by ndt80Δ after an 8-h induction. ChIP was performed as described previously by Yu and Koshland (2005) with anti-Pds5 and anti-HA antibodies. Note that Rec8 and Pds5 are not enriched at CARL2, which serves as a negative control. Error bars indicate SD.