Asunto(s)
Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Melanoma/metabolismo , Antígenos de Neoplasias/metabolismo , Diferenciación Celular , Análisis por Conglomerados , Femenino , Regulación Neoplásica de la Expresión Génica , Antígenos HLA/metabolismo , Humanos , Masculino , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas ras/metabolismoRESUMEN
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.
Asunto(s)
Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Biología Computacional/métodos , Fosfatos de Fosfatidilinositol/química , Proteómica/métodos , Línea Celular Tumoral , Citosol/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Liposomas/química , Espectrometría de Masas/métodos , Modelos Químicos , Fosfatos/química , Fosfatos de Fosfatidilinositol/metabolismo , Mapeo de Interacción de Proteínas , ProteomaRESUMEN
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.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfatidilinositol 4,5-Difosfato/química , Fosfatos de Fosfatidilinositol/química , Proteómica/métodos , Línea Celular Tumoral , Cromatografía por Intercambio Iónico/métodos , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Liposomas/química , Espectroscopía de Resonancia Magnética , Péptidos/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolípidos/química , Fosforilación , ProteomaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Química Farmacéutica/métodos , Imidazoles/síntesis química , Imidazoles/farmacología , Animales , Línea Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Imidazoles/química , Concentración 50 Inhibidora , Ratones , Modelos Químicos , Poríferos , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
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.