Figure 3.

AKT is involved in NLRP3 oligomerization and degradation with inflammasome activation.

(A) Immunoblots of DSS cross-linked insoluble fraction of DMSO- or MK2206-treated BMDMs after treating with the indicated stimuli: MK2206 (100 nM, pre-treated 1 h), LPS (1 μg/ml, 3 hrs) and Nigericin (5 μM, 15 min).

(B) Immunofluorescence images of BMDMs treated with indicated stimuli: MK2206 (100 nM, pre-treated 1 h), LPS (1 μg/ml, 3 hrs) and Nigericin (5 μM, 15 min), labeled with anti-ASC antibodies (green). The nuclei were labeled by DAPI (blue). Arrows indicate the ASC speck-like structure in the cells.

(C) Qualification of the percentage of the BMDMs with ASC speck like structure from (B). Experiments were repeated three times, more than 100 cells were qualified for each replicate, and bars represent the means ± SEM. **p<0.01, as determined by student’s t test.

(D) Immunoblots of the whole cell lysates of control-siRNA or AKT1/2-siRNA transfected THP-1 cells treated with the indicated stimuli: LPS (100 ng/ml, 6 hrs), Nig. (5 μM, 45 min). Expressions of endogenous NLRP3 protein level were normalized to untreated group.

(E) Immunoblots of the whole cell lysates of Flag-NLRP3 WT, S5A, or S5E overexpressed in control-siRNA transfected or AKT 1/2 depleted HEK293T cells. Blotted NLRP3 protein levels were normalized to control-siRNA transfected samples, respectively. Immunoblots shown are representative of at least 3 independent experiments and band quantification is shown for the representative blot.