Fig 2: SHP2 mediated the ubiquitination of TLR7 via dephosphorylating its Y1024 Immunoblotting of TLR7 ubiquitination in HEK293T cells transfected with GFP-TLR7 for 48 h, then treated with IMQ (10 µg/ml) or R848 (10 µg/ml) for 1 h.Immunoblotting of TLR7 ubiquitination in HEK293T cells co-transfected with GFP-TLR7, along with HA-Ub or mutant Ubs and treated with IMQ (10 µg/ml) for 1 h.Immunoblotting of HEK293T cells transfected with GFP-TLR7 and treated with or without IMQ (10 µg/ml) for 30 min.Immunoblotting of TLR7 ubiquitination in THP1 cells with vector, SHP2, shRNA-Control, or shRNA-SHP2 lentivirus was treated with IMQ (10 µg/ml) for 1 h.HEK293T cells co-transfected with GFP-TLR7 and HA-SHP2 and HA-SHP2 mutant vectors for 48 h, and then infected with IMQ (10 µg/ml) for 1 h.Immunoblotting of HEK293T cells transfected with GFP-TLR7 and TLR7 mutant vectors and treated with or without R848 (10 µg/ml) for 30 min.Immunoblotting of HEK293T cells transfected with GFP-TLR7 and TLR7 mutant vectors and treated with IMQ (10 µg/ml) for 30 min.Immunoblotting of HEK293T cells transfected with GFP-TLR7 and TLR7 mutant vectors and treated with or without IMQ (10 µg/ml).Immunoblotting of TLR7 ubiquitination in HEK293T cells with vector and SHP2 lentivirus transfected with GFP-TLR7 and GFP-TLR7Y1024D plasmids and infected with IMQ (10 µg/ml) for 1 h.Immunoblotting of HEK293T cells with shRNA-Control or shRNA-SHP2 lentivirus transfected with GFP-TLR7 and GFP-TLR7Y1024A plasmids and infected with IMQ (10 µg/ml) for 1 h. Source data are available online for this figure.

Fig 3: SHP2 interacted with TLR7 and promoted TLR7 trafficking to endosomes AHEK293T cells were transfected with plasmids expressing HA-tagged SHP2 and GFP-tagged TLR7. After 48 h, cells were left unstimulated or stimulated by IMQ (10 µg/ml) for 30 min. Cell lysates were immunoprecipitated by anti-HA or anti-GFP and probed by indicated antibodies.BPMA-differentiated THP-1 cells were unstimulated or stimulated by IMQ (10 µg/ml) for 30 min. Cell lysates were immunoprecipitated by anti-TLR7 or anti-SHP2 and probed by indicated antibodies.CBMDMs derived from wild-type mice were stimulated by IMQ (10 µg/ml) for 30 min. Cell lysates were immunoprecipitated by anti-SHP2 and probed by indicated antibodies.DRepresentative images of confocal microscopy performed with PMA-differentiated THP-1 cells treated with IMQ (10 µg/ml) for indicated durations. Scale bar: 20 µm.EImmunoblotting of HEK293T cells co-transfected for 48 h with GFP-TLR7, plus HA-SHP2, HA-SHP2 mutant vectors, followed by immunoprecipitation with anti-GFP beads.FPMA-differentiated THP-1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 (10 µg/ml). Subcellular fractionation was performed, and cytoplasmic and cell membrane proteins were probed by respective antibodies.G, HImmunoblot analysis of TLR7 and SHP2 in the isolated ER, Golgi, and endosome from PMA-differentiated THP-1 cells with either SHP2 overexpressed (G) and SHP2 deficiency (H).ISkin sections from psoriatic patients and healthy controls were immune-stained for TLR7 together with a marker of the early endosome (EEA1) or late endosome/lysosome (LAMP1) prior to analysis by confocal microscopy, showing TLR7 localization in different organelles. Scale bar: 50 µm. Source data are available online for this figure.

Fig 4: SHP099 provided no benefit in the Tlr7-Y1025D mice treated with IMQ Tlr7-Y1025D mutant Tlr7 ki male mice (n = 5/group) were treated with 10 mg/kg SHP099 or vehicle for 4 days. APhenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 µm. Left: H&E staining data; right: statistical data (mean ± SEM).BClinical scores plotted with mean ± SEM.CBody weight change plotted with mean ± SEM.D, EQuantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines (D) and other psoriasis-related cytokines (E) in the dorsal skin. Results were normalized to Gapdh expression. Data information: Data are represented as mean ± SEM. P values are determined by two-tailed Student’s t-test (A, D, E) or Bonferroni multiple-comparison test (B, C). ns, not significant. Source data are available online for this figure.

Fig 5: IRGM interacts with DNA/RNA sensors and utilizes autophagy to degrade them so as to constrain IFN response AImmunoprecipitation (IP) analysis of the interaction between endogenous IRGM and endogenous RIG-I, cGAS, MDA5, TLR3, TLR4, TLR7, NLRC4, and AIM2 in THP-1 cell lysates. IgG (H), IgG heavy chain.B–DCo-immunoprecipitation (Co-IP) analysis of the interaction between (B) GFP-IRGM and Flag-RIG-I, (C) GFP-IRGM, and Flag-cGAS, (D) GFP-IRGM and Flag-TLR3 in HEK293T cell lysates. L.E, Long exposure of input blot. IgG (L), IgG Light chain; IgG (H), IgG heavy chain.EGST pulldown assay of in vitro translated and radiolabelled myc-tagged cGAS, RIG-I, and TLR3 with GST or GST-tagged IRGM.FWestern blot analysis with the lysates of HT-29 cells stably transfected with control or Flag-IRGM plasmid and probed with the indicated antibodies.G–IWestern blot analysis with lysates of HEK293T cells transiently expressing GFP or GFP-IRGM and (G) Flag-RIG-I or (H) Flag-cGAS or (I) or Flag-TLR3. Two biological replicates are shown.J–KThe qRT–PCR analysis with RNA isolated from (J) control and Flag-IRGM stable HT-29 cells (K) THP-1 cells transiently transfected with control or Flag-IRGM plasmid. n = 3 (biological replicates). Mean ± SD, *P < 0.05, Student's unpaired t-test.L–NLuciferase reporter assays in HEK293T cells transiently transfected with ISRE reporter plasmid and other plasmids as indicated. Mean ± SD, n = 3 (biological repeats), *P < 0.05, **P < 0.005, ***P < 0.0005, Student's unpaired t-test.OWestern blotting analysis with lysates of control and untreated or bafilomycin A1 (300 nM; 4 and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.PWestern blotting analysis with lysates of control and untreated or chloroquine (50 µM; 4 and 8 h) treated Flag-IRGM expressing HT29 stable cell lines.QThe qRT–PCR analysis with RNA isolated from untreated or bafilomycin A1 (300 nM; 4, 8, and 12 h) treated control or Flag-IRGM expressing HT-29 stable cells as indicated. Mean ± SD, n = 3 (biological replicates), *P < 0.05, **P < 0.005 Student's unpaired t-test.Data information: *Non-specific, #degradation products of full-length GFP-IRGM.Source data are available online for this figure.