Figure 4.

rGRA9C decreases the activation of NLRP3 inflammation by associating NLRP3. (A) Bacterially purified 6xHis-GRA9C and rGRA9C^Q200L were analyzed by Coomassie blue staining (left) or immunoblotting (IB) with αHis (right). (B) BMDMs were incubated with rGRA9C and rGRA9C^Q200L for the indicated times and concentrations and cell viability measured with MTT assay. (C) BMDMs were treated with rVehicle, rGRA9C, or rGRA9C^Q200L, and immunolabelled with αFlag (Alexa 586), αNLRP3 (Alexa 488) and DAPI and analyzed in three dimensions. (D,E) BMDMs were primed with LPS (100 ng mL⁻¹) and stimulated with ATP (5 mM) and rVehicle, rGRA9C or rGRA9C^Q200L at various concentrations (0.1, 1, or 10 μg/mL). Followed by IP with αHis or αNLRP3 and immunoblotted with αNLRP3, αASC or αHis (D) or IB with in supernatant (SN) with αIL-1β p17, αIL-18 p18, or αCasp1 p10 and WCL with αHis, αPro-IL-1β, αPro-IL-18, αPro-Casp1, or αActin (E). (F) BMDMs were primed with LPS (100 ng mL⁻¹) and stimulated with ATP (5 mM) or DSS (3%) and rVehicle, rGRA9C, or rGRA9C^Q200L. The level of cytokines is analyzed by ELISA for IL-1β, and IL-18. The data are representative of four independent experiments with similar results (A–E). (F) Significant differences (*** p < 0.001) compared with rVehicle-treated BMDMs. Full-length images of the blots presented in the Figure S1.