Determination of protein phosphorylation in FcεRI-activated human mast cells by immunoblot analysis requires protein extraction under denaturing conditions

The advent of activation state antibodies has greatly facilitated studies aimed at understanding the intracellular signaling cascade following occupancy and/or aggregation of surface receptors. As part of an ongoing study investigating the signal transduction cascade initiated following aggregation of the high affinity receptor for IgE (FcepsilonRI) in human mast cells, we observed substantial differences in responses monitored by these antibodies when cells were extracted either under nonreducing or reducing conditions. This was true even in the presence of high concentrations of protease inhibitors. Although the activation of some proteins such as those of the MAP kinase pathway appeared to be unaffected by the extraction conditions, other signals, including overall tyrosine phosphorylation and activation of phospholipase Cgamma(1), were substantially different. This was due to the significant proteolysis in samples extracted under nondenaturing conditions. When the signaling proteins were extracted rapidly under denaturing conditions, however, there was little evidence of proteolysis of the signaling proteins with a resulting improved recovery of signal. Thus, accurate determination of signaling events utilizing activation state-specific antibodies in human mast cells requires protein extraction under denaturing conditions. The data presented in this report would be applicable to other cell types where high concentrations of proteases are present. (C) 2002 Elsevier Science B.V. All rights reserved.