CCDC134 interacts and stabilizes the ER chaperone Gp96. (A and B) Immunoprecipitates (IP) and whole-cell extracts (WCE) from transfected HEK293T (A) or knockout CAL-1 cell lines (B) as indicated. (C) Schematic of wildtype Gp96 and deletion constructs (left panel) as well as immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T transfected as indicated (right panel). SP: signal peptide, pN: pre-N-terminal domain; ND: N-terminal domain, CR: charged linker region; MD: middle domain; CD: C-terminal domain; KDEL: ER retention motif; steady state (N217 red) and cryptic N-glycans acceptor sites are shown. (D) Immunoprecipitates (IP) and input using Flag-CCDC134 and Gp96-HA recombinant proteins. For the complex formation, Flag-CCDC134 and Gp96-HA were preincubated overnight at 4°C before to perform the immunoprecipitation assay. Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA; asterisks indicate IgG heavy or light chains. (E) Immunoblots of lysates from indicated knockout CAL-1 cells. Asterisks indicate a non-specific band. (F) IL-6 production of indicated knockout CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (G) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) from indicated CAL-1 cell lines. (H) Immunoblots from indicated knockout CAL-1 cells treated with NMS-873 (1, 5, and 10 μM) or vehicle DMSO for 8 h. In A–E, G, and H, data are representative of two independent experiments. In F, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F3.