Monitoring cell signaling pathways by quantitative flow cytometry.
As the signaling pathways involved in leukemogenesis are being elucidated, several proteins have emerged as potential targets for therapy. Downstream from those targets are numerous intracellular factors that are constantly modulated. Monitoring those factors could provide insight into the potential efficacy of therapies by predicting which patients will respond to them and by determining the optimal dosage that will inhibit the target protein. We describe a flow cytometry method for quantitation of total and phosphorylated intracellular proteins. Compared with Western blot analysis, this technique dramatically decreases time and labor while providing multiparameter information on specific cell populations. As an example, total and phosphorylated CRKL is quantitated. The methodology has the potential for widespread application in the monitoring of targeted therapy.
Chan
Huai En Huang
HE
Department of Hematopathology, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, USA.
Jilani
Iman
I
Chang
Richard
R
Albitar
Maher
M
D000918:Antibody Specificity; D002463:Cell Membrane Permeability; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000276:immunology / Q000473:pathology / Q000503:physiopathology*; D015398:Signal Transduction / Q000502:physiology*