Journal: The Journal of Cell Biology
Article Title: A Natural Hepatocyte Growth Factor/Scatter Factor Autocrine Loop in Myoblast Cells and the Effect of the Constitutive Met Kinase Activation on Myogenic Differentiation
Figure Lengend Snippet: Double ectopic expression of h- met and h-HGF/ SF is required for the constitutive activation of h- met kinase. ( A ) Northern blot analysis of RNA expression levels of h- met and h-HGF/SF in clone 33 selected from C2 h- met /h-HGF/SF double transfection. Exogenous transcripts are larger than endogenous ones. ( B ) The h- met receptor expressed by clone 33 is phosphorylated on tyrosine residues. Cells were lysed at two different culture passages and immunoprecipitated with anti–human met antibodies. Proteins were resolved by 8% reducing SDSPAGE and immunoblotted with anti–human met ( α-hmet ) or anti-phosphotyrosine ( α-PTyr ) antibodies. ( C ) Phase-contrast and anti-MHC immunofluorescence micrographs of clone 33 cells exposed for 48 h in differentiation medium containing 10% HS. ( D ) Cell lysates from C2 parental cells and three single h- met –expressing C2 (C2/ h- met ) clones were immunoprecipitated with anti–murine met antiserum (C2 cells) or with anti-human met antiserum (C2/h- met clones), respectively. The immunocomplexes were resolved by 8% reducing SDS-PAGE and immunoblotted with anti–human ( α-h-met ) or anti-phosphotyrosine mAbs ( α-PTyr ). The almost undetectable tyrosine phosphorylation exhibited by h- met in comparison with the endogenous receptor suggests that it is not efficiently activated by murine HGF/SF.
Article Snippet: Finally, the immunocomplexes were solubilized in boiling Laemmli buffer containing 5% β-mercaptoethanol, electrophoresed on 8–12% SDS polyacrylamide gels, and transferred onto nitrocellulose filters (Hybond C; Amersham Intl.) by the semidry blot method.
Techniques: Expressing, Activation Assay, Northern Blot, RNA Expression, Transfection, Immunoprecipitation, Immunofluorescence, Clone Assay, SDS Page