RESUMO
At any time in vitro or in vivo expressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples. Quantification via protein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms. Here, wild-type green fluorescent protein (GFPwt) was expressed in high amounts in vitro using the Rapid Translation System 500 based on Escherichia coli lysates. Fluorescent activity was determined in dependence of oxygen and compared to total protein levels. In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable via determination of fluorescence activity. A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining is described.
Assuntos
Fluorescência , Proteínas Luminescentes/análise , Proteínas Luminescentes/biossíntese , Corantes , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Biossíntese de Proteínas , Corantes de Rosanilina , Espectrometria de Fluorescência , Transcrição GênicaRESUMO
Embryonic stem (ES) cells are pluripotent descendants of the inner cell mass of blastocysts capable of differentiating into progenitor cells of most if not all tissues. The pluripotency of ES cells is maintained by leukemia inhibitory factor (LIF), a member of the family of interleukin-6-type cytokines. These cytokines activate Janus tyrosine kinases and signal transducer and activator of transcription factors (Stat) via the signalling receptor component gp130. Pluripotent ES1 cells proliferating in the presence of LIF were known from previous studies to contain Stat3 and Stat1 capable of transcriptional activation. Here we report that the level of tyrosine-phosphorylated Stat3 decreases rapidly during differentiation induced by treatment of ES1 cells either with retinoic acid (RA) or by withdrawal of LIF. In line with this finding, the DNA-binding activity of Stat3 decreased during differentiation. In contrast, Stat5 was absent from pluripotent proliferating ES cells, but appeared early after induction of differentiation. The positive correlation between induction of differentiation and expression of Stat5 mRNA was confirmed for three independent ES cell lines. Stat5 transcripts were detectable in ES1 cells as early as 12 h after treatment with RA and 36 h after withdrawal of LIF. Stat5 protein was detectable 2 days after the onset of differentiation. These results establish Stat5 as a novel marker of very early stages of differentiation of ES cells.
Assuntos
Blastocisto/química , Proteínas de Ligação a DNA/análise , Interleucina-6 , Proteínas do Leite , Células-Tronco/química , Transativadores/análise , Animais , Biomarcadores , Blastocisto/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Cricetinae , Regulação para Baixo , Inibidores do Crescimento/fisiologia , Fator Inibidor de Leucemia , Linfocinas/fisiologia , Camundongos , Ligação Proteica , Fator de Transcrição STAT5 , Células-Tronco/citologiaRESUMO
Interleukin-6 (IL-6)-type cytokines activate transcription factors Stat1 and Stat3 (signal transducers and activators of transcription). Here we report that leukemia inhibitory factor (LIF) and IL-6 activate Stat5a in M1 myeloid leukemia cells in addition. In murine embryonal stem (ES) cells stably transfected with an expression vector for Stat5a treatment with LIF resulted in tyrosine phosphorylation and DNA-binding of this transcription factor. Transfection of an expression construct for Stat5a in human hepatoma cells caused a dose-dependent increase in LIF-triggered transcriptional activity. Our data demonstrate that Stat5a is activated by IL-6-type cytokines and can mediate transcriptional activity in addition to Stat1 and Stat3.
