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C2C12 myoblasts were cultured in GM containing fetal bovine serum (FBS, 10%) in Dulbecco's modified Eagle's medium (DMEM) with antibiotics and were transferred to DM containing FBS (0.1%) in DMEM plus insulin (5 μg/ml), transferrin (5 μg/ml), and antibiotics.
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32P]ATP for 30 min at 3O°C. Reactions were terminated by the addition of sample buffer, and GST-c-Jun was resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE), excised, and counted by liquid scintillation.
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note
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Approximately 50 to 100 μg of total cell lysate prepared as described was resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Membranes were blocked in nonfat dry milk (5%) and immunoblotted with either anti-MyoD (5.8A), anti-myogenin (F5D), anti-myosin heavy chain (MF20), anti-p21, anti-cyclin D1 (Transduction Laboratories, 1 μg/ml), or anti-p27 (Transduction Laboratories, 1 μg/ml) antibodies. Primary antibodies in nonfat dry milk (5%) were incubated with PVDF membranes for 2 to 4 hours at room temperature or overnight at 4°C. Proteins were visualized with either sheep antibody to mouse immunoglobulin G (lgG) or donkey antibody to rabbit lgG peroxidase-linked secondary antibodies (Amersham) at a dilution of 1:4000 and were developed with Enhanced Chemiluminescence (Amersham).
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note
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Myc-epitope-tagged MKP-1 (encoding amino acids 1 through 314, MKP-1-Myc) was subcloned into the retroviral tetracycline-repressible vector pBPSTR1 to generate pBPSTR1MKP1myc, which was transiently transfected by standard calcium phosphate precipitation into BOSC 23 cells in the presence of tetracycline (2 μg/ml). Viral supematants were collected 2 days later and were used to infect parental C2C12 myoblasts. Clones were isolated by selection in puromycin (1.5 μg/ml) in the presence of tetracycline (2 μg/ml). To induce expression of MKP-1-Myc, we trypsinized cells and washed them twice with GM. C2MKP-1 myoblasts were then replated in either GM or DM in the absence of tetracycline. Anti-serum to MKP-1 (Ab332) was generated by immunization of rabbits with purified mouse MKP-1 encoding residues 1 through 315 and was used at a 1:4000 dilution for immunoblot analysis. Cell lysates were prepared as described (9), and MKP-1 was immunoprecipitated with anti-MKP-1 (Ab332, 5 μl) followed by immunoblot analysis with either anti-MKP-1 (Ab332, 1:4000) or antibodies to Myc (9E10).
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note
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NIH 3T3 cells were transfected with the human cyclin D1 promoter fused to the luciferase gene (1 μg) and β-galactosidase (1 μg) with either wild-type [pEXV3.MEK(WT)] (4 μg) or constitutively active MEK [pEXV3.MEK(EE)] (4 μg) and pSG5.MKP-1-Myc (4 μg). We prepared lysates 2 days after transfection, and luciferase activity was measured on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, Ml) and β-galactosidase activity was assayed according to the manufacturer's protocol. Results are expressed as relative luciferase units normalized to β-galactosidase activity.
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35
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14444268781
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note
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NIH 3T3 cells were cotransfected with plasmids encoding MyoD (0.5 μg), rabbit muscle creatine kinase (MCK) promoter/enhancer luciferase reporter gene (1 μg), and β-galactosidase (1 μg), with either vector control, MEK(WT), or MEK(EE) (3 μg). We harvested cells 2 days after transfection and measured the luciferase and β-galactosidase activities. Expression of MEK(EE) inhibited MyoD transcriptional activity from the MCK luciferase reporter gene by ∼50% relative to MEK(WT).
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36
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14444275328
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note
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We thank W. Wright, P. Houghton, G. Hannon, D. Beach, R. Pestell, K. Walsh, and S. Reeves for reagents and T. Tiganis and M. Gutch for critical reading of this manuscript. Supported by a grant from The Council for Tobacco Research, USA, and a postdoctoral fellowship to A.M.B. from the Cold Spring Harbor Laboratory Association.
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