AIM2 Antibody - #DF3514

Figure 4 Rg1 inhibits pyroptosis through STAT3 signaling. (A) Conserved sequences at binding sites of STAT3. (B) STAT3 binding sites on AIM2 promoters predicted by JASPAR. (C) The binding relationship between STAT3 and AIM2 promoter verified by ChIP-qPCR experiment. *p < 0.05 compared with IgG negative control. (D) Western blotting analysis showing NLRP3, ASC, AIM2, cleaved-caspase-1, pro-caspase-1, IL-1β, mature-IL-1β, GSDMD and GSDMD-N protein expression in BV-2 cells with or without Rg1 pretreatment exposed to LPS or LPS with IL-6 stimulation, and with or without stattic treatment induced by LPS. (E–M) Bar graph shows gray value analysis based on immunoblot images. *p < 0.05, **p < 0.01, ***p < 0.001 compared with control, #p < 0.05 and ###p < 0.001 compared with LPS treatment group, ∆p < 0.05 and ∆∆∆p < 0.001 compared with LPS+Rg1 treatment group. (N) Flow cytometry shows the percentage of dead cells over total cells in each stage. The effect of Rg1 (60μM) in down-regulating the rate of apoptotic cells induced by LPS was abrogated by IL-6. Compared with LPS (2μg/mL) treatment group, the rate of apoptotic cells was markedly decreased in stattic and LPS co-incubated group. (O) Bar graph shows the statistical results of the rate of dead cells in each group. **p < 0.01 compared with control, ##p < 0.01 compared with LPS treatment group, ∆∆p < 0.01 compared with LPS+Rg1 treatment group.

Figure 3 Calpeptin inhibits AIM2 and NLRP3 inflammasome-mediated inflammation, and upregulates Klotho protein expression in IR mouse model. (A) Western blotting of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, pro-Caspase 1, cleaved-Caspase 1, IL-1β and IL-18 in the kidney of all mice. (B) Quantitative determination of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, cleaved-Caspase 1 and IL-18. (C) The mRNA levels of Calpain 2, Klotho, AIM2, ASC and GSDMD in the kidney among different groups. (D) The calpain activity of renal issues was measured by the relative fluorescence units (400/505 nm). (E) The cathepsin B activity of kidney issues was tested by the relative fluorescence units (400/505 nm). (F) Representative immunohistochemical micrographs from the kidney issues of different groups stained with Calpain 1, Calpain 2 and AIM2. ×400, bar = 50 μm. All data were presented as mean ± SEM (n = 3). NS, no significance; *p < 0.05 vs. sham group; **p < 0.01 vs. sham group; ***p < 0.001 vs. sham group; #p < 0.05 vs. IR group; ##p < 0.01 vs. IR group; ###p < 0.001 vs. IR group. CP, calpeptin; IR, ischemia/reperfusion; RFU, relative fluorescence units.

Figure 3 Calpeptin inhibits AIM2 and NLRP3 inflammasome-mediated inflammation, and upregulates Klotho protein expression in IR mouse model. (A) Western blotting of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, pro-Caspase 1, cleaved-Caspase 1, IL-1β and IL-18 in the kidney of all mice. (B) Quantitative determination of Calpain 1, Calpain 2, Klotho, AIM2, NLRP3, cleaved-Caspase 1 and IL-18. (C) The mRNA levels of Calpain 2, Klotho, AIM2, ASC and GSDMD in the kidney among different groups. (D) The calpain activity of renal issues was measured by the relative fluorescence units (400/505 nm). (E) The cathepsin B activity of kidney issues was tested by the relative fluorescence units (400/505 nm). (F) Representative immunohistochemical micrographs from the kidney issues of different groups stained with Calpain 1, Calpain 2 and AIM2. ×400, bar = 50 μm. All data were presented as mean ± SEM (n = 3). NS, no significance; *p < 0.05 vs. sham group; **p < 0.01 vs. sham group; ***p < 0.001 vs. sham group; #p < 0.05 vs. IR group; ##p < 0.01 vs. IR group; ###p < 0.001 vs. IR group. CP, calpeptin; IR, ischemia/reperfusion; RFU, relative fluorescence units.

Figure 4 Effect of AICAR treatment on NLRP3, NLRP1, NLRC4, and AIM2 inflammasome components (A,C) Representative immunofluorescence staining images showing the expressions of NLRP3 and caspase-1 in the spinal dorsal horn of sham and CIBP rats. Scale bar: 20 μm. (B,D) Quantitative analysis of fluorescence intensity in (A) and (C). Data are expressed as the mean±SD ( n=3). * P