Nbp2 62766

Whole-cell lysates were prepared from cells lysed in 1× RIPA buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₄P₂O₇ and protease inhibitors). Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% milk and 0.1% Tween-20 in TBS for 1 h at room temperature. Membranes were incubated with the appropriate primary antibodies overnight at 4°C. Finally, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and detected using the Pierce ECL Western blotting substrate kit (Thermo Fisher Scientific) and the Odyssey Fc, Dual-Mode Imaging system (Li-COR). The detection of IL-1β in the culture medium was undertaken using the human IL-1β Simple Step ELISA kit from Abcam (ab100562) and mouse IL-1β Simple Step ELISA kit from Abcam (ab197742) according to the manufacturer's instructions. The detection of IL-33 from cell lysates was undertaken using the human IL-33 Simple Step ELISA kit from Abcam (ab223865) and mouse IL-33 Simple Step ELISA kit from Abcam (ab213475) according to the manufacturer's instructions. Human and mouse HMGB1 was detected from cell lysates using human HMGB1 (Novus Bio, NBP2-62766) and mouse HMGB1 (Novus Bio, NBP2-62767) ELISA kits.

Enzyme-linked immunosorbent assay (ELISA) was used for quantitative determination of IL-1α (#DLA50, R&D Systems, Bio-Techne), HMGB1 (#NBP2-62766, Novus Biologicals, supplied by Bio-Techne) and HSP90α (#NBP2-76448, Novus Biologicals) in ACS conditioned medium. Protocols for all ELISAs adhered to the manufacturer’s instructions and data was obtained using a FLUOstar OPTIMA plate reader by spectrophotometric detection at 450 nm as previously (Dunnill et al., 2020 (link)).