Figure 2: Periodic regulation of Ssu72 phosphatase activity during the cell cycle.

(a–d) HeLa cells were synchronized in the G1/S boundary by using the double-thymidine block, in the G2 phase by doxorubicin treatment and in mitosis by nocodazole treatment. (a) Cellular extracts from synchronized cells were immunoprecipitated with the anti-Ssu72 antibody and then analysed by immunoblotting. (b) Purified Ssu72 proteins were incubated in a phosphatase buffer containing 10 mM pNPP. **P<0.01 and ***P<0.001, Student’s t-test; Error bars represent s.d. values from three independent experiments. (c) Cellular extracts from synchronized Control HeLa and Ssu72 knockdown (Ssu72 KD) cells were immunoprecipitated with normal immunoglobulin G (IgG) or an anti-SA2 antibody and immunoprecipitates were analysed by immunoblotting with anti-phospho-serine and anti-SA2 antibodies. (d) Synchronized cells were separated into non-chromatin supernatant (Supt) and chromatin (Chro) fractions, resolved by SDS–PAGE, and immunoblotted with anti-SA2, anti-Erk1, anti-H3 and anti-Ssu72 antibodies.