Figure S4.
Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA (dox.-ind. CCDC134) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: SourceData FS4.

Mapping of CCDC134 requirement for regulation of Gp96 in the ER. (A) Doxycycline-inducible CCDC134 mRNA (dox.-ind. CCDC134) levels in indicated cells measured by qPCR. The primers were specifically designed to detect only the doxycycline-inducible construct, excluding any detection of the endogenous CCDC134 mrRNA. CAL-1 cells stably expressing the doxycycline-inducible CCDC134 construct (clone 1 or 2) were treated with the indicated concentration of doxycycline (Dox., 0–10 ng/ml) for 24 h. (B) AlphaFold structure prediction for human CCDC134 (Uniprot entry name: Q9H6E4 CC134_HUMAN). (C) Immunoblots of cell lysate treated with EndoH (H) or PNGase F (F) of indicated CAL-1 cells reconstituted with wildtype or deletion CCDC134 mutants. The Δ133–156 CCDC134 deletion mutant lacks the predicted N-linked glycosylated NQT sequon. Ev: empty vector. (D) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged wildtype (WT) or deletion mutant CCDC134 constructs. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (E) Representative confocal microscopy images of HeLa cells transfected with Flag-tagged Δ133–156 CCDC134 deletion mutant which lacks the predicted N-linked glycosylated NQT sequon (left panel) and quantification of the colocalization between Flag-CCDC134 (wildtype or Δ133–156 deletion mutant) and calreticulin (right panel). Data are expressed as colocalization score (coloc. score) and pooled from three independent experiments. Each dot represents analysis of a single field of view containing one to four transfected cells, and violins show the variation of individual dot across all experiments; P value = ns, two-tailed Mann–Whitney test. Green: anti-Flag; red: anti-Calreticulin; blue: DAPI. Scale bar: 5 μm. (F) Immunoblots of proteins from the (not precipitated) supernatant (SN) of indicated CAL-1 cells. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 24 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. (G) Immunoblots of indicated CAL-1 cells incubated for 48 h with supernatants (SN) of donor cells cultured for 24 h in Opti-MEM. Doxycycline-inducible CCDC134 cells (clone 1) were induced with doxycycline (5 ng/ml, Dox.) for 48 h. SH: strep-HA tag, ind. 134: doxycycline-inducible CCDC134. In A, data show mean ± SD of three independent experiments. In C, F, and G, data are representative of two independent experiments. Source data are available for this figure: SourceData FS4.

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