RESUMO
We report the development of a new mouse anti-titin monoclonal antibody, named MAb Tit1 5H1.1, using the synthetic peptide corresponding to an amino acid sequence in the A-band of the titin molecule as immunogen. In the human skeletal muscle, MAb Tit1 5H1.1 reveals a clearly striated staining pattern, reacting with the A-band of the sarcomere. Electrophoretic, immunoblotting, and amino acid sequence analyses with ESI-MS/MS of human skeletal muscle tissue proved the target antigen of MAb Tit1 5H1.1 to be titin. The antibody reacts with titin also in non-muscle cells, producing a punctate pattern in cytoplasm and the nucleus. The most striking finding was a clear reaction of MAb Tit1 5H1.1 with centrioles in all cell types investigated so far. Immunocytochemical co-localization study with ninein-specific antibodies confirmed that the target antigen of MAb Tit1 5H1.1 is a centriole-associated protein. Experiments of the inhibition of synthesis of titin using titin siRNA duplex for the destruction of titin mRNA have shown a decreased staining of centrioles by MAb Tit1 5H1.1 in non-muscle cells and support the proposal that the target antigen of MAb is indeed titin. We suggest this anti-titin monoclonal antibody could be a valuable tool in the study of titin function and its subcellular location, both in muscle and non-muscle cells.
Assuntos
Anticorpos Monoclonais/biossíntese , Células/metabolismo , Centríolos/metabolismo , Proteínas Musculares/imunologia , Proteínas Musculares/metabolismo , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Células/efeitos dos fármacos , Células Cultivadas , Centríolos/efeitos dos fármacos , Conectina , Epitopos , Feminino , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Proteínas Quinases/genética , RNA Interferente Pequeno/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We report on the development of a mouse monoclonal antibody (named F10H2.B3) using the native cellular fragments of human fetal neural stem cells as immunogens. Molecular analysis has shown that the target antigen of F10H2.B3 is Ku80 (ATP-dependent DNA helicase 2 subunit 2 [EC 3.6.1.-]). We suggest this antibody could be used in certain conditions as a proliferation marker for cells of different origin.