Analysis of signaling events by dynamic phosphoflow cytometry.
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.
Firaguay
Guylène
G
1Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, F-13009 Marseille, France.
Nunès
Jacques A
JA
D000906:Antibodies; D002138:Calibration; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics; D004364:Pharmaceutical Preparations; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020298:U937 Cells; D014638:Vanadates / Q000494:pharmacology
D000906:Antibodies; D004364:Pharmaceutical Preparations; D010750:Phosphoproteins; D011506:Proteins; C076715:pervanadate; D014638:Vanadates; D011494:Protein Kinases; D051059:PTEN Phosphohydrolase