Fig 2: CMTM4 regulates IL-17RC plasma membrane localization.a, b, Immunoblotting analysis of CMTM4 and actin and flow cytometry analysis of IL-17RC surface expression in wild-type or several Cmtm4-/- ST2 cell lines (a) or Cmtm4-/- ST2 cell line #4 transduced with the expression vector coding for Myc-CMTM4 or EV (b). c, Real-time quantitative PCR analysis of the relative level of Cmtm4 transcript in wild-type and four Cmtm4-/- ST2 cell lines. Mean + s.e.m. from five independent experiments. Two-tailed Mann–Whitney test. d, Flow cytometry analysis of IL-17RA, IL-17RD, or TNFR1 surface expression in wild-type and Cmtm4-/- ST2 cells. e, Immunoblotting of IL-17RC, CMTM4, and actin, and flow cytometry analysis of IL-17RC surface expression in wild-type and Cmtm4-/- ST2 cells expressing IL-17RCTmRA. f-h, Immunoblotting analysis of CMTM4 and actin and flow cytometry analysis of IL-17RC and IL-17RA surface expression in wild-type or CMTM4-/- HeLa cells (f), CMTM4-/- HeLa transduced with the expression vector coding for Myc-CMTM4 or EV (g), and A549 wild-type, CMTM4-/- or control cell lines where CRISPR/Cas9 transfection did not cause CMTM4 deficiency (h). *, unspecific band. Flow cytometry data are based on five (e, f), four (d) or three (a, b, d, g, h) independent experiments. Mean + s.e.m., two-tailed unpaired t-test. n.s. not significant. MFI, median fluorescence intensity. Source data

Fig 3: CMTM4 is constitutively associated with IL-17RC transmembrane domain.a, MS analysis of IL-17RSC isolated via tandem affinity purification from lysates of ST2 cells that were stimulated with SF-IL-17A (500 ng ml–1) for 15 min. Proteins identified in stimulated samples, but not in control samples in which SF-IL-17A was added after lysis, are listed. Relative quantification is based on iBAQ values from 13 experiments. b, Immunoblotting analysis of samples isolated by Flag IP from lysates of ST2 cells transduced with expression vectors coding for SF-IL-17RA, SF-IL-17RC or EV. c,d, Immunoblotting analysis of samples isolated by Flag IP from lysates of ST2 cells expressing EV or SF-IL-17RC (c) or SF-IL-17RA (d) that were stimulated with IL-17A (500 ng ml–1) for 15 min or left unstimulated. *, unspecific band. e, Immunoblotting analysis of samples isolated through IP with IL-17RC antibody or isotype control antibody from the lysates of ST2 cells stimulated with IL-17A (500 ng ml–1) for 15 min or left unstimulated. f, MS analysis of proteins isolated via tandem affinity purification from the lysates of ST2 cells expressing SF-IL-17RC. Relative quantification is based on iBAQ values from three experiments. Only proteins that were not detected in control samples isolated from ST2 cells expressing SF-EGFP and that had an average iBAQ value >2 × 106 are listed. g, Mean + s.e.m. of iBAQ ratio between CMTM4 and IL-17RC in IL-17RSC calculated for individual MS experiments shown in a. h, Immunoblotting analysis of samples isolated by Flag IP from lysates of Drosophila S2 cells transduced with expression vectors coding for SF-IL-17RA, SF-IL-17RC or EV together with the expression vector coding for Myc-CMTM4. i, Immunoblotting analysis of samples isolated through Flag IP from lysates of ST2 cells transduced with expression vectors coding for SF-IL-17RA, SF-IL-17RC or indicated chimeric proteins. j, Immunoblotting analysis of samples isolated by Flag IP from lysates of ST2 cells expressing CMTM4-Myc, CMTM6-Myc or indicated chimeric proteins together with SF-IL-17RC. Data are representative of two (d,i,j), three (c,h) or four (b,e) independent experiments.Source data

Fig 4: CMTM4 is specifically associated with IL-17RC.a, b, Flow cytometry analysis of ST2 cells incubated with the indicated concentration of SF-IL-17A. Ligand binding was detected by Flag-APC antibody (a). Relative SF-IL-17A binding to ST2 cells was calculated from three independent experiments, mean ± s.e.m. (b). c, Immunoblotting analysis of lysates of wild-type ST2 cells that were stimulated with indicated concentrations of SF-IL-17A or commercial IL-17A (R&D Systems) for 15 min. d, Immunoblotting analysis of samples isolated through Flag immunoprecipitation from lysates of ST2 cells expressing SF-IL-17RB, SF-IL-17RD, or SF-IL-17RE. e, Comparison between murine and human IL-17RA and IL-17RC transmembrane domains. f, Flow cytometry analysis of surface expression of SF-IL-17RCWT and SF-IL-17RCTmRA ectopically expressed in ST2 cells. Mean + s.e.m. from six independent experiments. Two-tailed Mann–Whitney test. n.s. not significant. MFI, median fluorescence intensity. Data are representative of three (a–d) independent experiments. Source data

Fig 5: CMTM4 mediates IL-17RC glycosylation.a, Immunoblotting analysis of samples isolated via immunoprecipitation with IL-17RC antibody or control isotype antibody from lysates of wild-type or Cmtm4-/- ST2 cells. b,c, Immunoblotting analysis of samples isolated via immunoprecipitation with IL-17RC antibody from lysates of wild-type or several Cmtm4-/- ST2 cell lines (b) or Cmtm4-/- ST2 cells expressing Myc-CMTM4 or EV (c). d, Immunoblotting analysis of samples isolated via immunoprecipitation with IL-17RC antibody from lysates of wild-type or Cmtm4-/- ST2 cells treated or not with proteasome inhibitor bortezomib (10 nM) for 24 h. e, Immunoblotting analysis of samples isolated by Flag immunoprecipitation from lysates of Cmtm4-/- ST2 cells transduced with expression vectors coding for indicated Myc-tagged members of CMTM family together with the expression vector coding for SF-IL-17RC. f-j, Immunoblotting analysis of samples isolated by Flag immunoprecipitation from lysates of wild-type or Cmtm4-/- ST2 cells expressing SF-IL-17RA (f), SF-IL-17RB (g), SF-IL-17RD (h), SF-IL-17RE (i), or SF-TNFR1 (j) and treated or not with PNGase F. *, unspecific band. Data are representative of four (a), three (b–d), or two (e–j) independent experiments. Source data