Assuntos
Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Melanoma/metabolismo , Antígenos de Neoplasias/metabolismo , Diferenciação Celular , Análise por Conglomerados , Feminino , Regulação Neoplásica da Expressão Gênica , Antígenos HLA/metabolismo , Humanos , Masculino , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas ras/metabolismoRESUMO
Immobilizing chemically synthesized analogues of PI(3,4,5)P3 onto Affi-10 beads and incorporating them into liposomes allowed their use as affinity absorbents in the comprehensive analysis of the phosphoinositide interactome using cytosolic cell extracts of the LIM1215 colon cancer cell line. This led to the identification of 282 proteins that either interact with PI(3,4,5)P3 or are indirectly captured as part of a complex containing a PI(3,4,5)P3 binding partner. Identification of the proteins was achieved using affinity/LC-MS/MS experiments.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Biologia Computacional/métodos , Fosfatos de Fosfatidilinositol/química , Proteômica/métodos , Linhagem Celular Tumoral , Citosol/metabolismo , Glutationa Transferase/metabolismo , Humanos , Lipossomos/química , Espectrometria de Massas/métodos , Modelos Químicos , Fosfatos/química , Fosfatos de Fosfatidilinositol/metabolismo , Mapeamento de Interação de Proteínas , ProteomaRESUMO
A comprehensive analysis of the phosphoinositide interactome has been performed using analogues of PI(3,5)P2 and PI(4,5)P2 phosphatidyl phospholipids which were immobilized onto Affi-10 beads or incorporated into liposomes for use as affinity absorbents with cytosolic extracts from colonic carcinoma cell lines. Affinity/LC/MS/MS experiments allowed identification of 388 proteins/protein complexes that appeared to interact specifically with the phosphoinositide targets: a number of novel potential phosphoinositide interacting proteins have been identified.
Assuntos
Regulação Enzimológica da Expressão Gênica , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/química , Proteômica/métodos , Linhagem Celular Tumoral , Cromatografia por Troca Iônica/métodos , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Lipossomos/química , Espectroscopia de Ressonância Magnética , Peptídeos/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipídeos/química , Fosforilação , ProteomaRESUMO
A small series of derivatives of the alkaloid naamidine A was synthesized and tested in vitro for their ability to inhibit mitogenesis in BaF/ERX cells. Replacement of the imidazole core with a thiazole was found to have only a minor effect on potency, and the 4-methoxybenzyl substituent of the natural product was shown to be unnecessary for activity.
Assuntos
Antineoplásicos/farmacologia , Química Farmacêutica/métodos , Imidazóis/síntese química , Imidazóis/farmacologia , Animais , Linhagem Celular , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Imidazóis/química , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Poríferos , Relação Estrutura-Atividade , Tiazóis/químicaRESUMO
Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.
