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. 2011 Jul 4;39(18):7961–7973. doi: 10.1093/nar/gkr549

Figure 5.

Figure 5.

CKI isoform, CKIε associates with and phosphorylates Star–PAP. (A) In vitro kinase assay of the Flag–Star–PAP complex purified from HEK-293 cells stably expressing Flag–Star–PAP after knockdown of CKIα with specific RNAi or control RNAi followed by treatment with tBHQ (100 μM for 4 h) or DMSO using generic kinase substrate Casein. The 32P incorporated and the total protein (coomasie-stained) are indicated. (B) Western analysis to detect the association of various CKI isoforms as indicated in the Flag-purified Star–PAP complex from the cell in presence and absence of tBHQ treatment of the cell. Sup = Supernatant, F/T = Flow through, W = Wash, E = Elution fraction of the purification. (C) In vitro kinase assay of increasing amounts of CKIα with heat inactivated Flag purified Star–PAP as substrate. (D) In vitro kinase assay of increasing amounts of CKIε with heat inactivated cell-purified Flag–Star–PAP as substrate. (E) In vitro kinase assay of increasing amounts of CKIε with heat inactivated recombinant His-tagged Star–PAP as substrate. The 32P incorporated and the total protein (coomasie-stained) are indicated. (F) In vitro kinase assay of CKIα and CKIε with Flag–Star–PAP full length (FL) and deleted for proline rich region (ΔPRR) as substrate. The 32P incorporated, the total protein (coomasie-stained) and the immunoblot are indicated.