Article

Analysis of signaling events by dynamic phosphoflow cytometry.

1Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, F-13009 Marseille, France.
Science Signaling (Impact Factor: 7.65). 02/2009; 2(86):pl3. DOI: 10.1126/scisignal.286pl3
Source: PubMed

ABSTRACT Many proteins involved in cell signaling are phosphorylated. To determine the phosphorylation status of these signaling molecules at the single-cell level, we present a protocol for using state-specific antibodies to detect target phosphoproteins with fluorescence measurements by flow cytometry. To improve the signal intensity, a sandwich-labeling method for the analysis of signaling proteins is performed. By comparing the phosphorylation state of proteins in the presence and absence of sodium pervanadate, a nonspecific tyrosine phosphatase inhibitor, we determined the relative amount of tyrosine-phosphorylated protein in the samples, which reflects the activity of the signaling pathway. This dynamic approach, in combination with the signal amplification through a sandwich-labeling method, produces accurate and reproducible measurement of the activity of signaling pathways.

1 Follower
 · 
148 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described.
    BMC Cancer 08/2014; 14(1):565. DOI:10.1186/1471-2407-14-565
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The CD28 costimulatory receptor has a pivotal role in T cell biology as this molecule amplifies T cell receptor (TCR) signals to provide an efficient immune T cell response. There is a large debate about how CD28 mediates these signals. Here, we designed a CD28 gene-targeted knock-in mouse strain lacking the cytoplasmic tail of CD28. As is the case in CD28-deficient (CD28 knock-out) mice, regulatory T cell homeostasis and T cell activation are altered in these CD28 knock-in mice. Unexpectedly, the presence of a CD28 molecule deprived of its cytoplasmic tail could partially induce some early activation events in T cells such as signaling events or expression of early activation markers. These results unravel a new mechanism of T cell costimulation by CD28, independent of its cytoplasmic tail.
    Cellular and Molecular Life Sciences CMLS 03/2015; DOI:10.1007/s00018-015-1873-7
  • [Show abstract] [Hide abstract]
    ABSTRACT: We previously described a checkpoint for allelic exclusion that occurs at the pre-B cell to immature B cell transition and is dependent upon the IgH intronic enhancer, Eμ. We now provide evidence that the breach in allelic exclusion associated with Eμ deletion results from decreased Igμ levels that make it difficult for emerging BCRs to reach the signaling threshold required for positive selection into the immature B cell compartment. We show that this compartment is smaller in mice carrying an Eμ-deficient, but functional, IgH allele (VHΔ(a)). Pre-B cells in such mice produce ∼ 50% wild-type levels of Igμ (mRNA and protein), and this is associated with diminished signals, as measured by phosphorylation of pre-BCR/BCR downstream signaling proteins. Providing Eμ-deficient mice with a preassembled VL gene led not only to a larger immature B cell compartment but also to a decrease in "double-producers," suggesting that H chain/L chain combinations with superior signaling properties can overcome the signaling defect associated with low Igμ-chain and can eliminate the selective advantage of "double-producers" that achieve higher Igμ-chain levels through expression of a second IgH allele. Finally, we found that "double-producers" in Eμ-deficient mice include a subpopulation with autoreactive BCRs. We infer that BCRs with IgH chain from the Eμ-deficient allele are ignored during negative selection owing to their comparatively low density. In summary, these studies show that Eμ's effect on IgH levels at the pre-B cell to immature B cell transition strongly influences allelic exclusion, the breadth of the mature BCR repertoire, and the emergence of autoimmune B cells.
    The Journal of Immunology 09/2013; 191(8). DOI:10.4049/jimmunol.1301858

Full-text (2 Sources)

Download
68 Downloads
Available from
May 15, 2014