Skip to main content
. 2010 Sep 3;29(20):3544–3557. doi: 10.1038/emboj.2010.217

Figure 6.

Figure 6

Ssu72 dephosphorylates SA2. (A) Purified GST-SA2 or GST-Rad21 was reacted with recombinant Cdk1/cyclin B1 or Plk1 in the presence of radio-unlabelled ATP. In vitro-phosphorylated GST-SA2 or GST-Rad21 was incubated with purified His-Ssu72. The eluted beads were immunoblotted with anti-phospho-threonine (Inline graphicThr), anti-phospho-serine (Inline graphicSer), anti-SA2 and anti-Ssu72 antibodies. (B) His-tagged C-terminal SA2 peptides (His C-SA2) were incubated with recombinant Cdk1/cyclin B1 in the presence of [γ32P]ATP, washed and then reacted with purified GST, GST-Ssu72 WT, GST-Ssu72 C12S or λ phosphatase (λ PPase). The graph shows the relative signal intensities of the radio-labelled His C-SA2 peptides. (C) Purified GST-Rad21 proteins were incubated with Plk1 proteins in the presence of [γ32P]ATP, washed and then reacted with purified GST, GST-Ssu72 WT, GST-Ssu72 C12S (2 or 4 μg/ml) or λ PPase. (D) Dephosphorylation of SA2 by Ssu72. Nocodazole-treated (100 ng/ml, 12 h) HeLa cells were lysed and immunoprecipitated with an anti-SA2 antibody, and the resulting SA2–cohesin complexes were incubated with purified GST-Ssu72 WT, GST-Ssu72 C12S MT (1 or 2 μg/ml) or λ PPase (control). The bound SA2 proteins were resolved by SDS–PAGE and detected using antibodies against phospho-serine (Inline graphicSer), phospho-threonine (Inline graphicThr), SA2 or Smc1. (E) HeLa-Con and HeLa-Ssu72 cells were cultured and treated with nocodazole for 4 h. Endogenous SA2 was immunoprecipitated from cell extracts using an anti-SA2 antibody or normal IgG (negative control), and the eluted SA2 complexes were immunoblotted with anti-SA2, anti-phospho-serine and anti-phospho-threonine antibodies. (F) SA2–cohesin complexes were isolated from HeLa-Con or HeLa-Ssu72 cell extracts using a polyclonal anti-SA2 antibody, and the SA2 immunocomplexes were analysed by two-dimensional SDS–PAGE followed by immunoblotting with a polyclonal anti-SA2 antibody. (G) Nocodazole-treated (100 ng/ml, 12 h) control and Ssu72-knockdown cells were lysed and immunoprecipitated with an anti-SA2 antibody, and the resulting SA2 complexes were immunoblotted with anti-SA2, anti-phospho-serine (Inline graphicSer) and anti-phospho-threonine (Inline graphicThr) antibodies.