In vitro kinase assay

An in vitro BRAF activation kinase assay was conducted according to the instructions provided by Millipore (Billerica, MA). Briefly, reactions were performed in the presence of 10 µCi [γ³²P] ATP with active MEK1 (1 µg), 4HD or XAG (0, 5, 10, or 20 µM) in 40 µL of reaction buffer (40 mM MOPS/NaOH pH 7.0, 1 mM EDTA, 10 mM MnCl₂, and 0.8 M ammonium sulphate) at 30°C for 30 min. Reactions were stopped by adding 10 µL protein loading buffer and the mixture was separated by SDS-PAGE. The relative amounts of incorporated radioactivity were assessed by autoradiography.

An in vitro PI3-K assay was conducted at 30°C for 10 min with 100 ng of PI-3K and different doses of 4HD, XAG or LY294002. Each sample was mixed with 20 µL of 0.5 mg/ml phosphatidylinositol as substrate at room temperature for 5 min and then incubated an additional 10 min at 30°C with reaction buffer (0.25 mM ATP containing 10 µCi [γ-³²P]ATP, 10 mM Tris-HCl (pH 7.6), and 60 mM MgCl₂). The reaction was stopped by adding 15 µl of 4 N HCl and 130 µL of chloroform: methanol (1:1). After vortexing and fractionation, the lower chloroform phase was spotted onto a silica gel plate (EMD Millipore Corp.) that was preheated at 110°C for 1 h. The resulting radiolabeled spots were visualized by autoradiography.

In vitro CDK4 and CDK6 activation kinase assays were conducted according to the instructions provided by SignalChem (Canada). Briefly, reactions were performed in the presence of 10 µCi [γ³²P] ATP with 2 µg Rb-C fusion protein (701–928 amino acid) from Cell Signaling Technology, 4HD or XAG (0, 10, or 20 µM) in reaction buffer at 30°C for 30 min. Reactions were stopped by adding 10 µL protein loading buffer and the mixture was separated by SDS-PAGE. The relative amounts of incorporated radioactivity were assessed by autoradiography.