Monoclonal antibodies identify a possible regulatory domain of MyoD1.
A panel of monoclonal antibodies (mAbs) to murine MyoD1 was generated. One set of mAbs is shown to react with epitope(s) in the cysteine/histidine-rich (C/H) region while another set is shown to react with epitope(s) in the C-terminal portion of MyoD1. One of the mAbs reactive with a C-terminal epitope sensitively detected MyoD1 in whole cell extracts by Western blotting. Time course studies of total protein accumulation during C2C12 myoblast differentiation revealed only subtle changes in the phosphorylation and quantity of MyoD1 protein present in C2C12 cells from induction to 120 hr after induction. These results suggest that modulation of MyoD1 protein or total phosphorylation levels is not tightly associated with the transition of undifferentiated myoblasts to differentiated myocytes. Monoclonal antibodies to the C-terminal epitope produced supershifted bands in gel retardation assays, indicating that these mAbs had no effect on DNA binding. Although the C/H region of MyoD1 does not participate in DNA binding, mAbs reactive with the C/H region neutralized this activity in gel retardation assays. These data suggest that the conserved C/H domain may serve to modulate MyoD1 DNA-binding activity by interacting with another regulator.
Cole
F
F
Department of Pathology, Mount Sinai School of Medicine, New York, New York 10029.
Timo
K S
KS
Kohtz
D S
DS
D000818:Animals; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000166:cytology* / Q000378:metabolism; D009154:Mutation; D017570:MyoD Protein; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D009687:Nuclear Proteins; D010750:Phosphoproteins; D004247:DNA