Figure 4.
CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F4.

CCDC134 modulates TLR7/9 signaling by regulating Gp96 hyperglycosylation. (A) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1 or 2) treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) Immunoblots of knockout CAL-1 cells stably expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) treated with doxycycline (Dox., 5 ng/ml) for 24 h. (C) IL-6 production of indicated knockout CAL-1 cells expressing a doxycycline-inducible CCDC134 (ind. 134) construct (clone 1) induced with 5 ng/ml of doxycycline (Dox.) for 17 h and followed by 24 h stimulation with R848 (5 μg/ml). (D) Immunoprecipitates (IP) and whole-cell extracts (WCE) from TLR7-V5-expressing CAL-1 knockout cell lines as indicated. Asterisks indicate a non-specific band. (E) Immunoblots of lysates from indicated CAL-1 cell lines. long exp.: long exposure. (F) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of indicated CAL-1 cells treated with R848 (5 µg/ml, for 0–1 h). (H) Immunoblots of cell lysates treated with EndoH (H) or PNGase F (F). N3Q Gp96 bears mutations at positions N445Q-N481Q-N502Q. (I) IL-6 production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). In A, B, D, E, G, and H, data are representative of two independent experiments. In C, F, and I, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F4.

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