FIG 10.

PP2A is required for dephosphorylation of SAF-A S59 in mitosis. HeLa cells were treated with 100 nM siRNA scrambled control or siRNA to PP2A catalytic subunit (PP2Ac, alpha and beta isoforms). Eighty hours after transfection, 40 ng/ml of nocodazole was added, and the cells were returned to the incubator for a further 16 h. Cells were harvested by mitotic shake-off and left to recover in fresh medium (without nocodazole) for the times indicated. (A) Fifty micrograms of extract from asynchronized cells (Asy) or from nocodazole-treated cells was run on 8% or 10% acrylamide SDS gels and probed for PP2Ac, phosphoserine 59 SAF-A, total SAF-A (mouse monoclonal, Abcam number 10297), and Ku80 as indicated. Bands corresponding to SAF-A phosphoserine 59 were quantitated and normalized to total Ku80. (B) Quantitation of the experiment. Results are representative of 3 separate experiments.