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. 2013 Jun 9;288(29):20955–20965. doi: 10.1074/jbc.M113.486746

FIGURE 2.

FIGURE 2.

The two CDK-containing Mediator complexes interact with PRMT5 and WDR77 in HeLa nuclear extracts. A, HF-CDK8- and HF-CDK19-containing Mediator complexes were purified from HeLa nuclear extracts expressing either HF-CDK8 or HF-CDK19, using an anti-FLAG M2 agarose column. Purified proteins were subjected to SDS-PAGE (4–15% gradient gel, Bio-Rad), and the gel was stained with silver. Lane 1, protein standard marker; lane 2, mock-purified fraction (Mock); lane 3, HF-CDK8-containing Mediator fraction (HF-CDK8); lane 4, HF-CDK19-containing Mediator fraction (HF-CDK19). B, Western blotting of affinity-purified HF-CDK8- and HF-CDK19-containing Mediator complexes. Each complex was detected using anti-HA and anti-MED17 antibodies. Lane 1, mock-purified fraction (Mock); lane 2, HF-CDK8-containing Mediator fraction (HF-CDK8); lane 3, HF-CDK19-containing Mediator fraction (HF-CDK19). C, the interactions of PRMT5 and WDR77 with HF-CDK8- and HF-CDK19-containing Mediator complexes were detected using anti-PRMT5 and anti-WDR77 antibodies, respectively. Lane 1, 3% of input HeLa nuclear extract; lane 2, mock-purified fraction (Mock); lane 3, HF-CDK8-containing Mediator fraction (HF-CDK8); lane 4, HF-CDK19-containing Mediator fraction (HF-CDK19). These complexes were purified from the nuclear extracts used in A. IP, immunoprecipitation.