FIGURE 2.

P2Y₁₁ receptors preferentially couple to G_q and drive cytokine production in an IL‐1 receptor‐dependent manner. (a) IL‐6 and IL‐8, which are targets of IL‐1 receptor signalling, were measured in cell culture supernatants 24 h after stimulation with P2Y₁₁ receptor agonists (ATPγS and NF546). The antagonist NF340 was used to confirm that agonist‐mediated responses were specific to stimulation of P2Y₁₁ receptors and inhibitors were used to examine G protein coupling of P2Y₁₁ receptors (G_q: YM‐254890; G_s: indirectly via adenylyl cyclase: SQ 22536). *P ≤ .05, significantly different as indicated; one‐way ANOVA. (b) P2RY11 cells were transfected with either control siRNA or IL1R1 siRNA for 72 h, with or without subsequent P2Y₁₁ stimulation (ATPγS at 30 μM, 24 h). The level of knockdown was quantified by measuring IL‐1R1 protein surface expression by flow cytometry (left panel). Isotype control MFIs were subtracted from each staining and mean values of MFIs were calculated (n = 5) (right panel). *P ≤ .05, significantly different as indicated; one‐way ANOVA. (c) P2RY11 cells were transfected with either control siRNA or IL1R1 siRNA for 72 h and then treated for 24 h with P2Y₁₁ agonist (ATPγS) in the presence or absence of either P2Y₁₁ antagonist NF340 or recombinant human IL‐1 receptor antagonist (IL‐1RA). IL‐6 and IL‐8 were measured in cell culture supernatants. Data are means ± SEM from five independent cell populations. *P ≤ .05, significantly different as indicated; one‐way ANOVA