a, Representative flow cytometry analysis showing the percentage of TNF-positive cells, measured by intracellular cytokine staining, of indicated RAW macrophage lines after stimulation with CpG-B (25 nM), R848 (10 ng ml−1), ssRNA (1 µg ml−1), poly(I:C) (20 µg ml−1), or LPS (10 ng ml−1). Shaded histograms show unstimulated controls. b, TNF production, measured by ELISA, from the indicated RAW macrophage lines after stimulation for 8 h with R848 (10 ng ml−1), CpG-B (25 nM) or LPS (50 ng ml−1). Data are mean of n = 4 biological replicates pooled from two independent experiments. P values determined by unpaired two-tailed Student’s t-test. c, TLR7 and TLR9 trafficking are normal in UNC93B1PKP but not in UNC93B1(D34A) RAW lines. Immunoblot of TLR7–HA and TLR9–HA from lysates of indicated RAW macrophage lines. FL, full-length. All data are representative of three independent experiments.