Figure 4

ATR-107 inhibition on IL-21 induced STAT3 phosphorylation correlates inversely with IL-21R occupancy, ex vivo (A, B). Receptor occupancy assays and pSTAT3 assays were performed side by side in T cells (A) and B cells (B). Briefly, whole blood samples were pre-incubated with ATR-107 for 15 minutes at 37°C, and stained with cell surface markers (CD4, CD19) for 15 minutes. Then either ATR-107-A647 or 10 ng/ml of IL-21 were added to the wells, and incubated at 37°C for another 15 minutes (for pSTAT3 assay) or 20 minutes (for receptor occupancy assay), followed by the addition of the Lyse/fix buffer to stop the reaction and to remove RBC. After centrifugation, the cell pellets were washed twice with PBS + 2% FBS. Samples were then collected with (for pSTAT3 assay) or without (for receptor occupancy assay) permeabilization and stained with pSTAT3 A647. Data shown are the means ± SE from 8 donors.