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. 2023 May 18;26(6):106929. doi: 10.1016/j.isci.2023.106929

Figure 4.

Figure 4

ORF8 induces pro-inflammatory cytokines through binding to NLRP3

(A) ORF8 induces IL1β production through a non-surface-receptor-mediated process. PBMCs were pre-incubated on ice with ORF8 (500 ng/mL) for 2 h to allow ORF8 to bind to its “surface receptors” followed by washing and incubating at 37°C to activate the cytokine production in the presence or absence of additional ORF8 (200 ng/mL). IL1β expression was measured by qPCR.

(B) ORF8 directly binds to NLRP3 but not TLR2 nor TRL4 in primary monocytes shown by affinity pulldown assay.

(C) Schematic drawing of NLRP3 deletion constructs for mapping ORF8 binding domains in NLPR3.

(D) Mapping NLRP3 domains that bind to ORF8 by affinity pulldown assay using ORF8-Strep-Tacin beads. Western images of total cell lysates (left) were used as expression controls and Western images of precipitated proteins (right) showing ORF8 efficiently binds the full-length as well as two NLRP3 deletion mutant proteins.