Figure 1.

CKI-mediated Star–PAP phosphorylation is induced by tBHQ. (A) In vitro kinase assay of the Flag–Star–PAP complex purified from HEK-293 cells stably expressing Flag–Star–PAP after treatment with tBHQ (100 μM for 4 h) or the solvent control, DMSO using Star–PAP, Casein and Myelin basic protein (MBP) as substrates. The ³²P incorporated and the total protein (coomasie-stained) are indicated. (B) Quantification of the kinase activity toward Star–PAP, Casein and MBP from the autoradiogram in A. The intensity in arbitrary unit is expressed relative to the tBHQ untreated samples. (C) In vitro kinase assay as in A of the Flag Star–PAP complex purified from HEK-293 cells stably expressing Flag Star–PAP after treatment with tBHQ (100 μM for 4 h) or the solvent control, DMSO with no exogenous substrate in presence of increasing amounts of PI4,5P₂ (0, 25, 50 and 100 μM). The ³²P incorporated, total protein and the immunoblot for Star–PAP are indicated. (D) In vitro kinase assay as in C but after the treatment with 100 μM of CKI7 or 100 µM IC261 prior to initiation of the kinase reaction. Control represents the untreated sample.