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. 2011 Apr 21;6(4):e18985. doi: 10.1371/journal.pone.0018985

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Ammosova et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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293T cells were transfected with vectors expressing Flag-tagged CDK9 WT (lanes 1, 2 and 5) or CDK9 S175A mutant (lanes 3 and 4) and treated at 48 hours posttransfection with 100 nM okadaic acid and (³²P) orthophosphate. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies and subjected to dephosphorylation by PP1 as indicated. Lane 5, immunoprecipitation with non-specific mouse IgG. The reactions were resolved on 10% SDS-PAGE and analyzed on Phosphor Imager and by immunoblotting with anti-CDK9 antibodies. On a lower panel quantitation of (³²P) phosphorylation of CDK9 is shown. Results are from a typical experiment of 2 performed.