Evaluation of p47phox phosphorylation in human neutrophils using phospho-specific antibodies

Superoxide anions production by neutrophils plays a key role in host defense against pathogens and in inflammation. The enzyme responsible for this process is called the NADPH oxidase. It is a multicomponent enzyme comprised of a membrane-bound flavocytochrome b558 and several cytosolic proteins (p47phox, p67phox, p40phox, and p21rac1/2). The phosphorylation of p47phox is essential for the activation of the complex in intact cells. Until recently, analysis of the phosphorylation of p47phox in neutrophils required radioactive labeling, which implied the use of high amount of radioactive ((32)P)-orthophosphoric acid, high number of cells, and protein recovery by immunoprecipitation. In this study, we describe a radioactive-free technique to analyze the phosphorylation of p47phox in cell lysates, based on the use of phospho-specific antibodies, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting. This technique could be used to quickly and easily study the phosphorylation of p47phox under different conditions, such as testing the effects of pharmacological agents in this process or assessing the activation status of neutrophils in situ.