Detection of JNK and p38 activation by flow cytometry analysis.
JNK and p38 protein kinases are involved in the signal transduction of apoptotic stimulus. JNK and p38 are activated by dual phosphorylation on threonine and tyrosine residues. Different techniques such as Western blotting (WB) and confocal microscopy analysis have been developed to detect the activation by using antibodies that recognize the phosphorylated forms of both enzymes. However, these techniques are time consuming, not quantitative, and dependent on subjective interpretation. Herein, we describe a flow cytometry-based analysis to detect JNK and p38 activation. Using human primary lymphocytes and Jurkat CD4(+) T cells stimulated with PMA/ionomycin, we demonstrate activation (phosphorylation) of JNK and p38, which is further confirmed by two additional established techniques (WB and confocal microscopy). Flow cytometry analysis is shown to be more sensitive than WB to detect JNK and p38 activation, which can be quantitated and enables us to study their activation within cell populations.
Bou
German
G
Department of Immunology, Mayo Clinic, Rochester, MN 55905, USA.
Villasis-Keever
Angelina
A
Paya
Carlos V
CV
D015153:Blotting, Western; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D019169:Jurkat Cells; D018613:Microscopy, Confocal / Q000379:methods; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases
D015759:Ionomycin; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate