a, Schematic of the tested loop 5 mutants of UNC93B1 and the relative TLR9 responses indicated in shades of grey; white indicates a response equivalent to wild type, and black indicates no response. Asterisks show human UNC93B1 SNPs that have been tested in f. b, A larger region in loop 5 of UNC93B1 mediates binding to TLR9. Immunoprecipitation of UNC93B1–Flag from macrophage lines expressing the indicated UNC93B1 mutants (spanning amino acids 267–284, and non-functional HR) followed by immunoblot of TLR9–HA. Data are representative of two independent experiments. c, Intracellular cytokine staining of TNF in macrophage lines shown in b after stimulation with CpG-B (25 nM), R848 (100 ng ml−1) or LPS (10 ng ml−1). Shaded histograms show unstimulated controls. Data are representative of three independent experiments. d, Schematics showing relative positions and sequence alignment (bottom) of swapped regions within the TLR9/3 chimaeras. Coloured regions indicate TLR3 sequences. NF-κB luciferase assay in HEK293T cells transiently transfected with the indicated TLR9 and UNC93B1 mutants and stimulated with CpG-B (200 nM) for 16 h. Data are normalized to UNC93B1-independent human IL-1β responses and expressed as luciferase fold change over unstimulated controls. Data are mean ± s.d., n = 3 biological replicates. P values determined by two-way ANOVA followed by a Sidak’s post-test comparing each TLR9 allele co-expressed with UNC93B1 WT versus S282A (TLR9WT: P < 0.0001, TLR9Mut1: P > 0.9999, TLR9Mut2: P < 0.0001, TLR9Mut3: P < 0.0001, TLR9Mut4: P = 0.0020, TLR9Mut5: P = 0.0171). Data are representative experiment of two independent repeats. e, TLR9 mutants that rescue signalling in the presence of UNC93B1(S282A) also show normal binding to UNC93B1(S282A). Haemagglutinin immunoprecipitation of the indicated TLR9 mutants transiently expressed in HEK293T cells stably expressing the indicated UNC93B1–Flag alleles, followed by immunoblot of UNC93B1–Flag. Data are representative of three independent experiments. f, Human UNC93B1 variants with SNPs in loop 5 show decreased TLR9 signalling. NF-κB luciferase assay in HEK293T cells expressing TLR9 or TLR7 and the indicated human UNC93B1–Flag variants and stimulated with CpG-B (250 nM) or R848 (250 ng ml−1) for 16 h, respectively. Data are normalized to Renilla expression and expressed as RLUs. Data are mean ± s.d., n = 3 biological replicates. P values are determined by one-way ANOVA followed by a Tukey’s post-test. For CpG-B stimulations: P = 0.0048 (WT versus G270S), P = 0.0113 (WT versus R277Q), P = 0.9994 (WT versus G283R). Data are representative of four independent experiments. For R848 stimulations: P = 0.2001 (WT versus G270S), P = 0.0002 (WT versus R277Q), P = 0.9933 (WT versus G283R). Data are representative of three independent repeats experiment. g, Expression levels of the UNC93B1 mutants used in f. *P < 0.05, **P < 0.01, ***P < 0.001.