Fig. 5. Mincle activation leads to pro-IL-1β processing and secretion in TH17 cells.
a–d Polarized TH17 cells from indicated murine strains were stimulated with β-glucosylceramide (0, 5, 50 μg/ml) for 12 h, followed by western blot analysis of supernatants and cell lysates with the indicated antibodies, density values measured using Image J for the representative blot shown, ND not detected. e Supernatants from a–d were harvested and IL-1β concentrations were determined by ELISA, n = 3 biological replicates. f TH17 cells were stimulated with β-glucosylceramide (50 μg/ml) for 12 h, caspase 8 Glo Assay reagent was added to the media for another 1 h, followed by analysis by luminescence, n = 3 biological replicates. g IL-1β concentrations were analyzed from the supernatant of polarized TH17 cells pretreated with caspase inhibitors (YVAD-fmk/caspase1 inhibitor, IETD-fmk/caspase 8 inhibitor) and stimulated with β-glucosylceramide, n = 3 biological replicates. h Cell lysates from TH17 treated with β-glucosylceramide (50 μg/ml) were subjected to immunoprecipitation with anti-ASC, followed by western analysis with the indicated antibodies, data is representative of three independent experiments. *p < 0.05, ***p < 0.001 (two-sided student’s t test for e–g). Data are represented as mean ± SD. Exact P values for asterisks (from left to right): e 0.00015 0.00024 0.0015 f 0.0011 0.00002 0.0039 g <0.0001 0.0002.