a, UNC93B1PKP does not alter TLR7 export rates. Pulse-chase analysis of TLR7 in RAW macrophages expressing wild-type UNC93B1 and UNC93B1PKP. Cell lysate was immunoprecipitated with haemagglutinin and subjected to a radiolabelled screen and immunoblot. The full-length and cleaved forms of TLR7 are indicated. Asterisk denotes nonspecific band. Data are representative of two independent experiments. b, UNC93B1PKP does not affect TLR7 trafficking to endosomes. Levels of TLR7, LAMP1 and calnexin in whole-cell lysates or lysates of purified phagosomes from the indicated RAW macrophage lines were measured by immunoblot. Representative of three independent experiments. c, Colocalization of TLR7–HA (red) and LAMP1 (green) in RAW macrophages expressing the indicated UNC93B1–Flag alleles on a Myd88−/− background using super-resolution structured illumination microscopy. Boxed areas are magnified. The plot shows quantification of the percentage of total TLR7 within LAMP1+ endosomes, with each dot representing an individual cell. Data are mean ± s.d. and pooled from two independent experiments. Scale bars, 10 µm. P values determined by unpaired two-tailed Student’s t-test. d, The subcellular localization of UNC93B1PKP is not altered relative to wild-type UNC93B1. Co-localization of UNC93B1–Flag (red) and LAMP1 (green) was measured using super-resolution structured illumination microscopy in UNC93B1-deficient RAW macrophages complemented with wild-type, PKP or H412R mutant UNC93B1. A representative cell is shown for each UNC93B1 allele. Boxed areas are magnified. The plot shows quantification of the percentage of total UNC93B1 within LAMP1+ endosomes, with each dot representing an individual cell. Data are mean ± s.d. and acquired in a single experiment. Scale bars, 10 µm. P values determined by unpaired two-tailed Student’s t-test.