Stimulation of wild type and T735 mutant forms of ADAM17 by IL-1ß and anisomycin.

<p>A) Wild type mEFs were transfected with alkaline phosphatase-tagged substrates to monitor the sheddase activity of ADAM17 (TGFα, TNFα, CD62L and IL-1R2) or ADAM10 (BTC) and stimulated with IL-1ß (5 ng/ml) for 30 minutes. The release of the AP-tagged ADAM10 or ADAM17 substrates into the culture supernatant was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031600#s2" target="_blank">materials and method</a>s. B) <i>Adam17−/−</i> primary mEFs were transfected with TGFα-AP and either HA-tagged or untagged forms of the catalytically inactive ADAM17-E406A (EA), wild type ADAM17 (WT), or ADAM17 carrying point mutations at residue T735 (T735A or T735D), as indicated. The amount of TGFα-AP released in 30 minutes from untreated cells (black bars) or from cells stimulated with IL-1ß (5 ng/ml, light grey bars) or anisomycin (1 µM, dark grey bars) was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031600#s2" target="_blank">materials and methods</a>. C) Western blots of untagged or HA-tagged forms of ADAM17 (EA, WT, T735A or T735D) expressed in <i>Adam17−/−</i> mEFs were probed with antibodies against the cytoplasmic domain of ADAM17 (top panel) or the HA-tag (middle panel), or against ADAM9 as a loading control. D) Shedding of transfected human TNFα from <i>Adam17−/−</i> mEFs rescued with WT or the T735A, T735D or EA mutant forms of ADAM17. The amount of TNFα released into the conditioned media from unstimulated cells or cells stimulated with IL-1β (5 ng/ml) in 30 minutes was determined by ELISA. All data represent the average of at least three separate experiments, and the error bars correspond to +/− standard error of the mean (sem). Asterisks indicate a significant increase upon addition of the stimulatory agent (students t-test, p<0.05). NS indicates that no significant difference was found between the indicated conditions.