Skepinone l

BMMCs, PCMCs or the MC/9‐NFAT and MC/9‐NF‐κB reporter MC lines ³⁸ were washed and seeded in IL‐3‐free media at a density of 1x10⁶ cells/mL. After 1 h, cells were treated with cyclosporine A (4 µg/ml) (CaN inhibitor, CaN‐i), the Ca²⁺ chelator BAPTA‐AM (5 µM) (Ca²⁺ chel.) for Ca²⁺ depletion, the IKK VII (1 µM) (IKK inhibitor, IKK‐i), U0126 (10 µM) (ERK1/2 inhibitor, ERK‐i) (all from Merck), the selective P2X7 inhibitor, A438079 (50 µM) (ATP‐antagonist, ATP‐ant.) (Sigma), skepinone‐L (1 µM) (p38 inhibitor, p38‐i) (Cayman) or respective amounts of the vehicles (DMSO) for 30 min. All used inhibitors did not influence cell viability. Subsequently, cells were stimulated with rmIL‐33 (50 ng/mL) (PeproTech) and/ or if not otherwise shown with ATP (500 µM) (Sigma). For Western blotting experiments, BMMCs were stimulated for different time points (as shown in the figures). For ELISA experiments, supernatants were collected after 24 h, and for degranulation assays (CD107α surface expression), MCs were stimulated for 30 min. To investigate the activation of NFAT or NF‐κB in the MC/9 reporter MC lines [38 (link)], we stimulated for 8h.

BMMCs (1 × 10⁶ cells/ml) were seeded in IL‐3‐ and serum‐free media. After 1 h, cells were pre‐treated with the inhibitors cyclosporine A (4 µg/ml) (CaN inhibitor, CaN‐i), the Ca²⁺ chelator BAPTA‐AM (5 µM) (Ca²⁺ chel.) for Ca²⁺ depletion, U0126 (10 µM) (ERK1/2 inhibitor, ERK‐i) (all from Merck) or skepinone‐L (1 µM) (Cayman) (p38 inhibitor, p38‐i) or vehicle for 30 min. Afterwards, BMMCs were stimulated with IL‐33 (50 ng/ml) (PeproTech) or ATP (500 µM) (Sigma) or both in combination for 24 h. Supernatants were transferred to pre‐coated ELISA plates and the competitive LTC₄ ELISA was performed as described in the instructions (Cayman).