Detection of intact transcription factors in human neutrophils.
The crucial contribution of neutrophils to innate immunity extends well beyond their traditional role as professional phagocytes. Indeed, it is now well established that neutrophils generate a plethora of inflammatory cytokines and chemokines that are profoundly involved in the onset and evolution of the inflammatory reaction. Several recent studies have also shown that neutrophils can represent an important source of inflammatory cytokines in a number of pathophysiological settings. The inflammatory cytokines produced by neutrophils are generally encoded by immediate-early response genes, which in turn depend on the activation of transcription factors such as those belonging to the nuclear factor (NF)-kappaB and signal transducers and activators of transcription (STAT) families. We have shown in the past that such factors are expressed and inducible in neutrophils stimulated by physiological agonists. However, the detection of intact (i.e., undegraded) transcription factors in neutrophils requires special precautions and an alternative protocol, as a result of the huge amounts of endogenous proteases present in these cells. This protocol is the focus of this chapter.
McDonald
Patrick P
PP
Department of Pharmacology, Univeristy of Illinois,Chicago, IL, USA.
Ye
Richard D
RD
D002457:Cell Extracts / Q000737:chemistry; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D002469:Cell Separation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006801:Humans; D009504:Neutrophils / Q000737:chemistry* / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D014157:Transcription Factors / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism
D002457:Cell Extracts; D010761:Phosphorus Radioisotopes; D014157:Transcription Factors