Journal:

Article Title: Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism

doi: 10.1038/sj.emboj.7600328

Figure Lengend Snippet: No requirement of 14-3-3 binding for the inhibitory effect of Thr504 phosphorylation. (A) Activated eggs expressing GST-Cdc25A proteins (together with or without Δ60-Chk1) as in Figure 4B were subjected to GST pulldown assays and analyzed by immunoblotting using anti-GST antibody or anti-human 14-3-3β antibody (which can recognize all 14-3-3 isoforms). (B) Chk1-phosphorylated GST-Cdc25A protein (wild type) was GST-pulled down from activated eggs, washed with or without 0.4% Empigen, and analyzed by immunoblotting as in (A) (left). Extracts from the eggs expressing GST-cyclin A1 (together with Xe-Wee1B; see Figure 4C legend) were treated with the indicated peptide-coupled beads and immunoblotted for cyclin A1 or 14-3-3 protein (right). See Materials and methods for details. (C) GST-Cdc25A proteins (phosphorylated or not by Chk1) and egg extracts (equivalent to 10 eggs) were both prepared as in (B) (GST-Cdc25A proteins being eluted from glutathione beads), mixed together, and incubated for 1 h at 4°C. GST-Cdc25A proteins were then immunoprecipitated with anti-Cdc25A antibody (IP) and analyzed by immunoblotting using anti-GST or anti-cyclin A1 antibodies. As control of GST-Cdc25A proteins (wild type or T504A), GST alone was used (Cont.). (D) GST-Cdc25A proteins prepared as in (C) were first incubated or not with 0.1 μg of recombinant Xenopus 14-3-3ɛ protein in 30 μl of an egg extraction buffer for 15 min at 4°C and then with (GST-)Cdk1–cyclin A1 complexes (purified from 10 eggs) for 1 h. (Xenopus 14-3-3ɛ protein can bind to phosphorylated Thr504 of Cdc25A; see Materials and methods.) The mixture was then analyzed as in (C). In (A–D), all the GST-Cdc25A constructs had both S73A and C428S mutations.

Article Snippet: Routinely, proteins equivalent to one egg or embryo were analyzed by immunoblotting ( Shimuta et al , 2002 ), using the above-described anti-phospho-Thr504 antibody, anti- Xenopus Cdc25A antibody ( Shimuta et al , 2002 ), anti-Myc antibody (A-14, Santa Cruz), anti-GST antibody (Z-5 or B-14, Santa Cruz), anti- Xenopus cyclin A1 or B1 antibodies (a gift from J Maller), anti- Xenopus cyclin E1 antibody (a gift from T Kishimoto), anti-Cdk1 phospho-Tyr15 antibody (♯911, Cell Signaling), anti-human Chk1 phospho-Ser345 antibody (♯2341, Cell Signaling), or anti-human 14-3-3β antibody (H8, Santa Cruz).

Techniques: Binding Assay, Expressing, Western Blot, Incubation, Immunoprecipitation, Recombinant, Purification, Construct