Figure 3.

Ser296-phosphorylated Chk1 binds 14-3-3γ. (A) In vitro kinase activity of individual immunoprecipitated Myc-Chk1 forms (WT or S296A) towards the GST-Cdc25C fragment (195-256 a.a.). Fold activation after UV irradiation is also indicated (mean±s.e.m. from three independent experiments). (B) Detection of endogenous 14-3-3γ in anti-Chk1 immunoprecipitates after UV irradiation. UCN-01 pre-treatment was performed as described in ‘Materials and methods'. (C) Effects of Chk1 mutation on the complex formation after UV irradiation. Myc-Chk1 WT, K38M or S296A was purified as an anti-Myc immunoprecipitate. (D, E) GST pull-down assays using GST-Chk1 purified from Sf9 cells and each subtype of 14-3-3 protein purified from bacteria. Each purified 14-3-3 protein was indicated in (D). A total of 0.4 μg of GST-Chk1-His was incubated in 20 μl of reaction mixture (25 mM Tris–HCl [pH 7.5], 10 mM MgCl₂) with (+) or without (−) 100 μM ATP at 30°C for 60 min. Then, the mixture was rotated with glutathione beads at 4°C. Beads were washed with IP buffer and then rotated with 0.4 μg of each purified 14-3-3 protein in 100 μl of IP buffer at 4°C for 1 h. Beads were then washed and subjected to immunoblotting with anti-pan-14-3-3 (characterized in Supplementary Figure S3A).