Journal: BMC Biochemistry

Article Title: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5

doi: 10.1186/1471-2091-6-27

Figure Lengend Snippet: Myf5 mitotic degradation is conserved in non-muscle cells. HeLa-S3 cells expressing Myf5 under an inducible promotor (tet off ) were treated (lanes 2, 3, 4) or not (AS, lane 1) with nocodazole (200 ng/ml) for 16 h, as in [20]. In lanes 3 and 4, cells were treated with a proteasome inhibitor (MG132, 50 μM) for 2 h prior to lysis (as described in Methods). In lane 4, extract was incubated with Lambda phosphatase (New England Biolabs) for 30 min at 30°C. 25 μg of each extract were resolved by 10% SDS-PAGE and immunoblotted with anti-Myf5 antibodies. AS: asynchronous cells; * indicates a non specific band recognized by the antibodies that can be used as a loading control.

Article Snippet: Ubiquitylation assays are performed in the presence of 200 μM MG132 (BioMol), 5 μM Ubiquitin aldehyde (BioMol), 1 mg/ml Ubiquitin (SIGMA-ALDRICH), 5 mM MgCl 2 and 1 mM ATPγS.

Techniques: Expressing, Lysis, Incubation, SDS Page, Control

Journal: BMC Biochemistry

Article Title: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5

doi: 10.1186/1471-2091-6-27

Figure Lengend Snippet: Myf5 is degraded in a cell-cycle dependent manner in Xenopus egg extracts, and its degradation is correlated with changes in its phosphorylation status. (A) Myf5 is phosphorylated and degraded in mitotic (CSF) extracts, but dephosphorylated and stable in interphase extracts. Degradation assays were performed as described in Methods; 2 μl samples were taken at the indicated times and resolved by 10% SDS-PAGE. (B) Degradation of Myf5 was quantified and normalized (start = 100%) : black squares represent the average degradation of Myf5 in interphase extracts (5 independent experiments), open circles the average degradation of Myf5 in CSF extracts (7 independent experiments), and black circles the average degradation of dephosphorylated (deP) Myf5 (see Methods section) in CSF extracts (4 independent experiments). Bars represent standard deviation. For each lane, a region corresponding to all non-phosphorylated and phosphorylated forms of Myf5 was quantified using the ImageQuant software (Molecular Dynamics). The apparent increase of Myf5 level in interphase extracts is probably due to the accumulation of all the initially phosphorylated forms of Myf5 (some of them being close to background) into the fastest migrating form of Myf5. (C) All the slower migrating forms of Myf5 are phosphorylated: lane 1: in vitro translated Myf5; lane 2: in vitro translated Myf5 after 1 h incubation with CSF extract in the presence of 1 μM microcystin LR (this treatment leads to hyperphosphorylation of Myf5 (Myf5 PPP ), see text and figure 3 for details); lanes 3–6: in vitro translated Myf5 (lanes 3 and 4), or Myf5 PPP (lanes 5 and 6) were immunoprecipitated with anti-Myf5 antibodies and incubated in the absence or presence of Lambda phosphatase (λ PPase), as indicated. Samples were resolved by 10% SDS-PAGE and analyzed using a PhosphoImager. (D) A mitotic extract was first incubated with 200 μM MG132, or the same volume of pure DMSO as a control, for 10 min at 25°C; dephosphorylated (deP) Myf5 (see method) was then added to the treated extract. At each time point, 2 μl samples were resolved by 10% SDS-PAGE. (E) Relative amounts of Myf5 in the gel shown in D were quantified as above and reported on the graph.

Article Snippet: Ubiquitylation assays are performed in the presence of 200 μM MG132 (BioMol), 5 μM Ubiquitin aldehyde (BioMol), 1 mg/ml Ubiquitin (SIGMA-ALDRICH), 5 mM MgCl 2 and 1 mM ATPγS.

Techniques: Phospho-proteomics, SDS Page, Standard Deviation, Software, In Vitro, Incubation, Immunoprecipitation, Control

Journal: BMC Biochemistry

Article Title: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5

doi: 10.1186/1471-2091-6-27

Figure Lengend Snippet: Hyperphosphorylated Myf5 is stable. An equal volume of reticulocyte lysate containing in vitro translated Myf5 and of Xenopus mitotic egg extract (CSF) were incubated together for 60 min in the presence of 1 μM microcystin LR. At each time point, 0.5 μl samples were analyzed by 10% SDS-PAGE (panel A). After this initial incubation, 2 μl of the reaction mixture containing hyperphosphorylated Myf5 (Myf5 PPP ) were further incubated in 18 μl of fresh mitotic extract, either in the presence of 1 μM microcystin (panel B) or without inhibitor addition (panel C), or without microcystin and in the presence of the proteasome inhibitor MG132 (200 μM, panel D). At each time point, 2 μl samples were analyzed by 10% SDS-PAGE.

Article Snippet: Ubiquitylation assays are performed in the presence of 200 μM MG132 (BioMol), 5 μM Ubiquitin aldehyde (BioMol), 1 mg/ml Ubiquitin (SIGMA-ALDRICH), 5 mM MgCl 2 and 1 mM ATPγS.

Techniques: In Vitro, Incubation, SDS Page

Journal: BMC Biochemistry

Article Title: Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5

doi: 10.1186/1471-2091-6-27

Figure Lengend Snippet: Polyubiquitylation of Myf5 is required for its degradation. (A) Myf5 is polyubiquitylated in mitotic extracts: Myf5 was incubated in a mitotic extract in the presence of ubiquitin (1 mg/ml), MG132 (200 μM) and ubiquitin aldehyde (5 μM). At the times indicated, 2 μl samples were analyzed by 10% SDS-PAGE to visualize the formation of high-MW adducts ((Ub) n -Myf5, poly-ubiquitylated Myf5 molecules). (B) Lysine-less (K0) ubiquitin inhibits Myf5 polyubiquitylation: Myf5 ubiquitylation was performed in a mitotic extract in the presence of MG132 (200 μM), ubiquitin aldehyde (1 μM), ubiquitin (Ub) and lysine-less-ubiquitin (UbK0) at the indicated concentrations, for 30 min at 25°C. (C) Lysine-less ubiquitin stabilizes Myf5: Degradation assays were performed in 20 μl reaction mixtures containing 16 μl of mitotic extract, 2 μl of radiolabeled in vitro translated Myf5 and a mixture of wild type and K0 ubiquitin as indicated. The reaction was performed at 25°C for 40 min, 2 μl samples were resolved by 10% SDS-PAGE. "START" corresponds to a 2 μl sample taken at time 0 from the reaction containing 1 mg/ml Ub. Radioactivity was quantified as previously described (see legend of figure 2) and percentages of degradation were calculated for each Ub/UbK0 ratio from the difference between the times 0 (not shown) and 40 minutes.

Article Snippet: Ubiquitylation assays are performed in the presence of 200 μM MG132 (BioMol), 5 μM Ubiquitin aldehyde (BioMol), 1 mg/ml Ubiquitin (SIGMA-ALDRICH), 5 mM MgCl 2 and 1 mM ATPγS.

Techniques: Incubation, Ubiquitin Proteomics, SDS Page, In Vitro, Radioactivity