Fig 2: IL-33 is inactivated by disulphide bonding.(a) Concentration of IL-33 (mean±s.e.m.) in bronchoalveolar lavage fluid (BALF) following intranasal Alternaria (ALT) challenge of BALB/c mice (n=3 per group), representative of 2 independent studies. (b) Western blot analysis of the same samples (pooled per group) under reducing and non-reducing conditions. Controls are as follows: cell lysate, lysate of HEK cells transfected with full length mouse IL-33; R&D, truncated mouse IL-33 (R&D systems); pep, truncated mouse IL-33 (Peprotech); PBS (30 min), BALF from vehicle (PBS) challenged mice at 30 min timepoint. (c) Non-reduced SDS-PAGE of IL-33112–270 either untreated (-) or post treatment with cell culture media (Iscoves Modified Dulbeccos Media) (+). Monomeric IL-33 was purified prior to analysis. (d) The human IL-33 linear sequence illustrated with the position of the cysteines and the proposed disulphide bridges formed after media treatment. Disulphide mapping was performed on purified monomeric protein. Numbering is based on the full length IL-33 sequence. (e) Hydrogen-exchange mass spectrometry (HX-MS) analysis of monomeric IL-33 and DSB IL-33. Comparison of fractional hydrogen exchange (for deuterium) in IL-33 (left panel) and DSB IL-33 (right panel). Data are mapped onto the published IL-33 structure22 in both cases for comparison purposes. Gaps in sequence coverage where no HX-MS data could be obtained are highlighted in slate blue. Side chains of cysteine residues are displayed as sticks. (f) Structural model displaying the difference in fractional hydrogen exchange between IL-33 and DSB IL-33 overlaid with the ST2 binding sites (red and magenta)23. Only increased hydrogen exchange was observed for DSB IL-33 versus IL-33, with regions exhibiting the greatest differences of hydrogen exchange depicted in dark blue. (g) ST2 binding of human IL-33 (upper panel) and purified DSB IL-33 (lower panel) measured by surface plasmon resonance. (h) Signalling in human umbilical vein endothelial cells (HUVEC) stimulated by human IL-33 and purified DSB IL-33. NF?B p65/RelA translocation was measured at 30 min post stimulation. Data points are mean±s.e.m. of duplicate determinations, representative of 3 independent experiments.