Figure 2.
Loss of the ER-resident protein CCDC134 impaired endolysosomal TLR7/9 maturation. (A) Schematic of wildtype, deletion, or tagged CCDC134 constructs. L: linker, SH: Strep-HA tag. (B) Representative confocal microscopy images of HeLa cells transfected with wildtype or Δ2–25 mutant CCDC134 (left panel) and quantification of the colocalization between CCDC134 and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents the analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dots across all experiments; P value <0.0001, two-tailed Mann–Whitney test. Green: anti-CCDC134; red: anti-Calreticulin; blue: DAPI. Scale bar: 10 μm. (C) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F) from HEK293T cells transfected as indicated. (D) Immunoblots of proteins precipitated from supernatant (SN) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (E) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (F–H) Immunoblots of lysates from indicated CAL-1 cells untreated (F) or treated with EndoH (H) or PNGase F (F) (G and H). Red arrows indicate the EndoH-resistant full-length form of TLR9. Ev: empty vector, gF: glycosylated full-length; dgF: deglycosylated full-length; gC: glycosylated cleaved form; dgC: deglycosylated cleaved form; long exp.: long exposure. In C, D, and F–H, data are representative of two independent experiments. In E, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F2.

Loss of the ER-resident protein CCDC134 impaired endolysosomal TLR7/9 maturation. (A) Schematic of wildtype, deletion, or tagged CCDC134 constructs. L: linker, SH: Strep-HA tag. (B) Representative confocal microscopy images of HeLa cells transfected with wildtype or Δ2–25 mutant CCDC134 (left panel) and quantification of the colocalization between CCDC134 and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents the analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dots across all experiments; P value <0.0001, two-tailed Mann–Whitney test. Green: anti-CCDC134; red: anti-Calreticulin; blue: DAPI. Scale bar: 10 μm. (C) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F) from HEK293T cells transfected as indicated. (D) Immunoblots of proteins precipitated from supernatant (SN) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (E) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (F–H) Immunoblots of lysates from indicated CAL-1 cells untreated (F) or treated with EndoH (H) or PNGase F (F) (G and H). Red arrows indicate the EndoH-resistant full-length form of TLR9. Ev: empty vector, gF: glycosylated full-length; dgF: deglycosylated full-length; gC: glycosylated cleaved form; dgC: deglycosylated cleaved form; long exp.: long exposure. In C, D, and F–H, data are representative of two independent experiments. In E, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F2.

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