Phospho-NLRP3 (Ser194) Antibody - #AF3555

Figure 4.5-HT/HTR3A enhances NLRP3 inflammasome activation by facilitating inflammasome assembling. A–C, mRNA expression of NLRP3, IL1B, and IL18 detected by real-time PCR in THP-1 cells after activation with LPS (100 ng/mL, 4 hours), ATP (5 mmol/L, 1 hour) with/without TPS (100 μmol/L), or 2Met5HT (100 nmol/L) for 5 hours. D, Coimmunoprecipitation of NLRP3 inflammasome components [pro–caspase-1 (P-casp1), NLRP3, and ASC] with anti-ASC from lysates. G, ASC oligomerization analysis for THP-1 cells (with/without anti-HTR3A shRNA transfection) after activation with LPS (100 ng/mL, 4 hours) and ATP (5 mmol/L, 1 hour) with exogenous 5-HT (10 μmol/L, 5 hours) treatment. Coimmunoprecipitation of NLRP3 inflammasome components (P-casp1, NLRP3, and ASC) with anti-ASC from lysates (E) and ASC oligomerization analysis of THP-1 cells (H) after activation with LPS (100 ng/mL, 4 hours) and ATP (5 mmol/L, 1 hour) with/without exogenous 5-HT (10 μmol/L) and TPS (100 μmol/L) treatments for 5 hours. Coimmunoprecipitation of NLRP3 inflammasome components (P-casp1, NLRP3, and ASC) with anti-ASC from lysates (F) and ASC oligomerization analysis of THP-1 cells (I) after activation with LPS (100 ng/mL, 4 hours) and ATP (5 mmol/L, 1 hour) with/without 2Met5HT (100 nmol/L) treatment for 5 hours. J, Representative photos of ASC speck staining in THP-1 and iBMDM cells after activation with LPS (100 ng/mL, 4 hours) and ATP (5 mmol/L, 1 hour) with/without 5-HT(10 μmol/L) plus TPS (100 μmol/L) or 2Met5HT (100 nmol/L) treatments for 5 hours. A–C, Data represent mean ± SD for at least three independent experiments; one-way ANOVA. **, P < 0.01. n.s., no significant differences. Ctrl, control.

Figure 2 STS inhibits the activation of NLRP3 inflammasome in diabetic aortas, HG-treated ECs and VSMCs. (A,B) Representative immunofluorescence images (400 × , scale bar: 20 μm) and summarized integrated density of NLRP3 and ASC colocalization in aortas; (C,D) Representative Western blot gels and summarized data show the production of NLRP3 dimer, the expression of NLRP3, the phosphorylation of NLRP3 at serine 194 site in aortas; (E–G) The summarized data show active caspase-1, mature IL-1β and IL-18 in aortas; (H,I) Representative Western blot gels and summarized data show the production of NLRP3 dimer, the expression of NLRP3, the phosphorylation of NLRP3 at serine 194 site in ECs; (J–L) The summarized data show the active caspase-1, mature IL-1β and IL-18 in ECs; (M,N) Representative immunofluorescence images (800 × , scale bar: 10 μm) and summarized integrated density of NLRP3 and ASC colocalization in ECs; (O,P) Representative Western blot gels and summarized data show the production of NLRP3 dimer, the expression of NLRP3, the phosphorylation of NLRP3 at serine 194 site in VSMCs; (Q–S) The summarized data show the active caspase-1, mature IL-1β and IL-18 in VSMCs; (T,U) Representative immunofluorescence images (800 × , scale bar: 10 μm) and summarized integrated density of NLRP3 and ASC colocalization in VSMCs. *P