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Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the "analog-sensitive" mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.

We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other noncommercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a wellperforming inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested.

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