The possibility that reversing the resulting Michael addition and thiocarbamate products with DTT might result in identification of the targets of these reactions using PROP. Some evidence suggests that this is not so. For example, modification of the MEKK1 protein kinase by isothiocyanate is stable to boiling in SDS and DTT, though other cysteine adducts might be less stable. Similarly, numerous cell proteins labeled with biotin-prostaglandin J2, and detected with anti-biotin immunoblotting are not measurably reversed by boiling in SDS and DTT, though again, specific proteins might be. Owing to these observations, we predict that these reactions will not be detectable using PROP. We and others have previously detected regulatory oxidation of other protein kinases. Importantly, in most of these experimental systems, oxidation is induced by hydrogen peroxide at high concentrations, diamide, menadione, or other non-physiologic agents. Our finding that micromolar concentrations of PGJ2 can induce oxidation and inhibition of p38 extends these results to agents involved in the physiologic response to disease or inflammation. Our results suggest that oxidation may serve as a redox switch that provides a feedback mechanism for p38 and other signaling kinases. Specifically, initial MK-2206 2HCl activation of p38 results from phosphorylation within the “activation loop”, that is detectable using anti-phospho p38 antibodies. Subsequently, activated p38 may become oxidized at one or more cysteine residues, resulting in the loss of kinase activity. Importantly, this inactive kinase retains its phosphorylation and may incorrectly be interpreted as an active kinase when using anti-phospho antibody immunoblot as a surrogate measure of “activity”. Signal transduction investigators are cautioned that the simplicity of an anti-phospho-immunoblot may obscure more subtle means by which signal transduction is regulated. We recommend that for a specific signaling pathway to be deemed “activated”, a direct measurement of the catalytic activity in vitro is indicated, together with an evaluation of natural substrates of the kinase within cells. Gap junction channels are formed when connexin subunits situated in adjacent plasma membranes dock together. These transmembrane channels allow the passage of metabolites, ions and second messengers and nucleotides up to 1 kDa in size. Undocked connexins, or hemichannels have also been identified and they provide a means to contact the extracellular environment. Gap junction channels are formed when connexin subunits situated in adjacent plasma membranes dock together.