CCDC134 controls Gp96 protein glycosylation and stability. (A) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (B) Volcano plot of proteins, identified by mass spectrometry, from Flag-immunoprecipitates from sgCCDC134 CAL-1 stably reconstituted with Flag-CCDC134 or control CAL-1 (sgRen transduced with empty vector [EV]). red: fold change (FC) >2; gray: fold change (FC) = [−2, 2]; blue: fold change (FC) less than −2. (C) Immunoprecipitates (IP) and whole-cell extracts (WCE) from HEK293T cells transfected as indicated. (D) Coomassie blue staining of recombinant proteins Gp96-HA or Flag-CCDC134 (Flag-134) in a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Red arrow indicates specific Gp96-HA or Flag-CCDC134 bands; black arrow indicates putative C-terminal species of Gp96-HA. (E) Immunoblots of indicated knockout HEK293T cells. (F) Immunoblots of indicated CAL-1 cells. (G)HSP90B1 (gene coding for Gp96) mRNA levels of indicated CAL-1 cells measured by qPCR (normalized to HPRT1). (H) Immunoblots of indicated knockout CAL-1 cells treated with tunicamycin (Tun.) (5 µg/ml, for 0–4 h) or vehicle DMSO. In A, C–F, and H, data are representative of two independent experiments. In G, data show mean ± SD of four independent experiments. Source data are available for this figure: SourceData FS3.