Fig. 6: Competition between Ripk2 and ASC for Caspase-1 interaction.
From: Card9 protects sepsis by regulating Ripk2-mediated activation of NLRP3 inflammasome in macrophages
A BMDMs were transfected with si-Ctrl or si-Ripk2, mRNA expression was detected using qPCR and protein level was detected using western blotting. B, C BMDMs were transfected with siRNA-Ctrl or siRNA-Ripk2-3. cells were unstimulated (medium) or pretreatment with LPS (100 ng/ml) for 3 h, and then treated with ATP (5 mM, 1 h). B ELISA analysis of IL-1β and IL-18 secretion in the culture supernatant. C Western blot analysis of cell lysates of BMDMs. D, E BMDMs were transfected with Ripk2 over-expression plasmid, cells were unstimulated (medium) or pretreatment with LPS (100 ng/ml) for 3 h, and then treated with ATP (5 mM, 1 h). D ELISA analysis of IL-1β and IL-18 secretion in the culture supernatant. E Western blot analysis of cell lysates of BMDMs. F Differential Caspase-1 interactions upon LPS + ATP stimulation. BMDMs containing V5-Caspase-1 were stimulated with LPS + ATP for the indicated times, followed by IP with anti-V5 and IB with anti-ASC or anti-Ripk2. G Competition between ASC and Ripk2 for Caspase-1 interaction. HEK293T cells post-transfection with V5-Caspase-1 or Flag-ASC together with increasing amounts of HA-Ripk2 for 48 h. Then, anti-V5 was used for IP and anti-Flag, anti-HA, or anti-V5 were used for IB. Data are shown as mean ± SEM. Each group was representative of at least three biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.
