Fig. 3. NPC1 is a lysosomal adaptor that mediates STING degradation.

a, Immunoblot analysis of the STING signalling cascade. Npc1^WT and Npc1^KO MEFs were stimulated with the STING agonist DMXAA (30 μg ml⁻¹) for 0, 1, 3, 5 or 7 h. The total and phosphorylated proteins immunoblotted are identified on the left. b, c, STING and NPC1 interaction domain mapping. HEK293T cells were transfected with the indicated plasmids (top), and 24 h later, the indicated antibodies were used for the pull-down. b, Full-length (FL) HA–NPC1 co-immunoprecipitated with Flag–STING truncated variants. c, Full-length HA–STING co-immunoprecipitated with Flag–NPC1 truncated variants. EV, empty vector; IP, immunoprecipitation. d–g, Cell-free lysosome recruitment assay. d, Diagram of the assay. e, Representative images of 6×His–STING–EGFP beads incubated with LysoCtrl–mCherry-, LAMP1–mCherry- and NPC1-mCherry-labelled lysosomes. f, Quantification of the recruitment of lysosomes to 6×His–STING–EGFP beads. For quantification, NPC1–mCherry, n = 33 beads; LAMP1–mCherry, n = 20 beads; LysoCtrl–mCherry, n = 20 beads. Data are mean ± s.d. Unpaired two-tailed Student’s t-test. g, Immunoblot analysis of the indicated proteins in different MEFs. Data are representative of at least two independent experiments.