Fig. 4: Als3-mediated IL-1β processing requires NLRP3, MLKL, and caspase-8.
From: Fungal Als proteins hijack host death effector domains to promote inflammasome signaling
a, b LPS-primed BMDMs were untransfected or transfected with Als3-His or Als3S170Y-His (5 μg ml−1), and caspase-1 (p10) and/or IL-1β (p17) processing was measured by immunoblotting. Profect-P2 transfection reagent was used for protein transfection. BMDMs were primed with 200 ng ml−1 LPS for 3 h. BMDMs from mouse mutants including Nlrp3-/- (a), Mlkl-/- (b), or Mlkl-/-Casp8-/- (b), were used together with WT. c, d GM-CSF cultured BMDMs were stimulated with the indicated agents for 2 days or LPS for 3 h. The levels of IL-1β and TNF in supernatants were quantitated by ELISA. Als3/Als3S170Y, 15 μg ml−1; Curdlan, 1 mg ml−1; LPS, 200 ng ml−1, Nec1s 10 µM, z-VAD-fmk 50 µM. Cell viability was normalized based on the viability of cells cultured with medium only. Data in (c, d) represent the mean ± s.e.m. of data from two independent experiments with two or three technical repeats (n = 5). P-values for the samples within the group with or without z-VAD-fmk were calculated with one-way ANOVA with Tukey post-hoc analysis. P-values for the samples between the groups with or without z-VAD-fmk were calculated with an unpaired two-tailed t test. The data presented in (a, b) are representative of three independent experiments.
