Journal: iScience

Article Title: Regulation of the endoplasmic reticulum stress sensor ATF6α through multiple oxidoreductases in the ER

doi: 10.1016/j.isci.2026.116290

Figure Lengend Snippet: ATF6α C467 dimerization is regulated by Trx2 domain of ERdj5 (A) Knockdown (KD) efficiency of ERdj5. HEK293T cells were transfected with ERdj5 siRNA and analyzed by immunoblotting with an anti-ERdj5 antibody under reducing conditions. Actin was used as a loading control. Original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study are contained in . (B) Formation of C467 dimers via ERdj5. HEK293T cells (wild-type; WT) and ERdj5-KD cells were treated with 1 μM Tg for the indicated periods and analyzed by immunoblotting with anti-ATF6α antibodies under nonreducing conditions. Representative immunoblots from three independent experiments are shown ( n = 3). (D) A series of ERdj5 mutants was constructed in this study. One of the CXXC motifs in the Trx domain of ERdj5 was replaced by SXXS. (E) C467 dimerization through ERdj5 mutants. HEK293T cells were transfected with FLAG-tagged WT or mutant ERdj5 and analyzed by immunoblotting with anti-ATF6α and anti-FLAG antibodies under reducing and nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (G) C467 dimerization through the ERdj5 C2/CC mutant. HEK293T cells were transfected with FLAG-tagged WT or mutant ERdj5 and analyzed by immunoblotting with anti-ATF6α and anti-FLAG antibodies under reducing and nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (I) Regulation of C467 dimerization by ERp18 and ERdj5. HEK293T cells were transfected with Myc-tagged ERdj5 and/or FLAG-tagged ERp18 and analyzed by immunoblotting with anti-ATF6α, anti-FLAG, and anti-Myc antibodies under reducing and nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (C, F, H, and J) The intensity of the C467 dimer bands, as shown in (B, E, G, and I). The bands were quantified using ImageJ software and normalized to the total intensity (monomer and C467 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The DOI for the Mendeley Data is listed in the . contains original uncropped western blot images used for quantification (n = 3 independent biological replicates), and contains original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study.

Techniques: Knockdown, Transfection, Western Blot, Control, Construct, Mutagenesis, Software, One-tailed Test

Journal: iScience

Article Title: Regulation of the endoplasmic reticulum stress sensor ATF6α through multiple oxidoreductases in the ER

doi: 10.1016/j.isci.2026.116290

Figure Lengend Snippet: Redox states of ERdj5 and ATF6α (A) PEG-maleimide assay. The redox state of proteins is fixed by trichloroacetic acid (TCA) precipitation. The thiol groups of cysteine residues are then modified with PEG-maleimide and detected by SDS-PAGE. This modification causes an upward band shift for proteins in the reduced state, whereas oxidized proteins do not shift. The oxidizing agent diphenyl disulfide (DPS) promotes disulfide bond formation and inhibits PEG-maleimide modification, while the reducing agent dithiothreitol (DTT) reduces disulfide bonds and enhances PEG-maleimide modification. (B) The redox state of ATF6α was determined by nonreducing SDS-PAGE. ATF6α-knockout (KO) cells were transfected with the p3×FLAG-tagged AAACC mutant and analyzed by immunoblotting with anti-FLAG antibodies under nonreducing conditions. The bands from top to bottom indicate the C467 dimer, C618 dimer, reduced monomer and oxidized monomer. Reduced monomer indicates the reduced form of ATF6α; oxidized monomer indicates the oxidized form of ATF6α. Original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study are contained in . (C) The redox state of the ERdj5 mutants was observed using PEG-maleimide. WT or ATF6α-KO cells were transfected with FLAG-tagged ERdj5 mutant and analyzed by immunoblotting with anti-ATF6α and anti-FLAG antibodies under reducing conditions. Representative immunoblots from three independent experiments are shown ( n = 3). (D) The intensity of reduced form bonds, as shown in (C). The bands were quantified using the ImageJ software and normalized to the total intensity (reduced form and oxidized form). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01). (E) ERdj5 oxidizes Cys467 of ATF6α via the Trx2 domain, and ATF6α forms the C467 dimer.

Article Snippet: The DOI for the Mendeley Data is listed in the . contains original uncropped western blot images used for quantification (n = 3 independent biological replicates), and contains original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study.

Techniques: TCA Precipitation, Modification, SDS Page, Electrophoretic Mobility Shift Assay, Knock-Out, Transfection, Mutagenesis, Western Blot, Software, One-tailed Test

Journal: iScience

Article Title: Regulation of the endoplasmic reticulum stress sensor ATF6α through multiple oxidoreductases in the ER

doi: 10.1016/j.isci.2026.116290

Figure Lengend Snippet: ERdj5 regulates ATF6α dimerization through dual function (A) Steps for substrate reduction by reductase and stabilization of the complex by the CA mutant. (B and C) Interaction between ERdj5 and ATF6α. HEK293T cells were transfected with FLAG-tagged WT or mutant ERdj5 and Myc-tagged WT or mutant ATF6α, and the cell lysates were prepared for immunoprecipitation with anti-FLAG. The immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies under reducing conditions. Original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study are contained in . (D) Reduction of C618 dimers by ERdj5. ATF6α-KO and ATF6α-KO ERdj5-KD cells were transfected with p3×FLAG-tagged WT ATF6α and analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies under reducing or nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (E) The intensity of each band, as shown in (D). The bands were quantified using the ImageJ software and normalized to the total intensity of each band position (monomer, C467 dimer, and C618 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The DOI for the Mendeley Data is listed in the . contains original uncropped western blot images used for quantification (n = 3 independent biological replicates), and contains original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study.

Techniques: Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Control, Software, One-tailed Test

Journal: iScience

Article Title: Regulation of the endoplasmic reticulum stress sensor ATF6α through multiple oxidoreductases in the ER

doi: 10.1016/j.isci.2026.116290

Figure Lengend Snippet: C618 dimers suppress C467 dimerization and its transport to the Golgi apparatus (A) Evaluation of ATF6α glycan levels. ATF6α-KO cells were transfected with p3×FLAG-tagged WT ATF6α and pretreated with 2 mM BADAP and 50 μM PF429242 (S1P inhibitor) for 1 h prior to the induction of ER stress with 5 μM Tg, and the results were analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. The cell lysates were left untreated or treated with Endo H. g and u indicate glycosylated and unglycosylated proteins, respectively. Original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study are contained in . (B) Transport and C467 dimerization of the ATF6α mutant during ER stress. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or C618A mutant ATF6α, pretreated with 50 μM PF429242 for 1 h prior to the induction of ER stress with 5 μM Tg for 0, 15, and 30 min, and analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (C) The intensity of the C467 dimer bands, as shown in (B). The bands were quantified using the ImageJ software and normalized to the total intensity (monomer, C618 dimer, and C467 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (D) The intensity of the C618 dimer bands, as shown in (B). The bands were quantified using ImageJ, normalized to the total intensity (monomer, C618 dimer, C467 dimer, and Golgi-C467 dimer), and plotted against time. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (E) The intensity of the Golgi-C467 dimers, as shown in (B). The bands were quantified using ImageJ, normalized to the total intensity (monomer, C618 dimer, C467 dimer, and Golgi-C467 dimer), and plotted against the time. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (F) Interaction between ATF6α and BiP. ATF6α-KO cells were transfected with p3×FLAG-tagged ATF6α and Myc-tagged BiP, and cell lysates were prepared for immunoprecipitation with an anti-Myc antibody. The immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies under nonreducing and reducing conditions. Representative immunoblots from three independent experiments are shown ( n = 3). (G) The intensity of each ATF6α band, as shown in (F). The bands were quantified using ImageJ software and normalized to the BiP (Input/IP) intensity. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (H) Transport of ATF6α mutant during ER stress. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or mutant ATF6α, pretreated with 50 μM PF429242 for 1 h prior to the induction of ER stress with 5 μM Tg for 1 h, and analyzed by immunoblotting with anti-FLAG antibodies under reducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (I) The intensity of the Golgi-ATF6α bands, as shown in (H). The bands were quantified using the ImageJ software and normalized to the total intensity (ATF6α-F and Golgi-ATF6α). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (J and K) Immunofluorescence of HeLa cells exogenously expressing GFP-tagged WT or mutant ATF6α. HeLa cells were transfected with N-terminal GFP-fused ATF6α (WT and various mutants). For ER localization analysis, cells were fixed and stained with calnexin (CNX), an ER-resident protein, without Tg treatment (J). For Golgi localization analysis, cells were pretreated with 50 μM PF429242 for 1 h and subsequently treated with 500 nM Tg for 30 min before staining with GM130, a Golgi marker (K). All cells were counterstained with Hoechst 33342 and immunostained with the indicated antibodies. Scale bars, 5 μm. (L) The percentage of cells showing colocalization between GFP-fused ATF6α and GM130, as shown in (K). The means ± standard error of the mean (SEM) of at three independent experiments, each performed with 10 cells. Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05). (M) ATF6α-KO cells were transfected with p3×FLAG-tagged WT or mutant ATF6α, pretreated with 5 μM Tg for 0, 30, and 60 min, and analyzed by immunoblotting with anti-FLAG antibodies under reducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (N) The intensity of ATF6α-N bands at 30 min after Tg treatment, as shown in (M). The bands were quantified using ImageJ, normalized to the total intensity (ATF6α-F and ATF6α-N). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05). (O) BiP mRNA levels in ATF6α-KO cells overexpressing WT or mutants under Tg treatment conditions. After treatment with 500 nM Tg for 8 h (+Tg), BiP mRNA levels were quantified by qPCR. The expression levels were normalized to GAPDH and compared to those in untreated cells (−Tg). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The DOI for the Mendeley Data is listed in the . contains original uncropped western blot images used for quantification (n = 3 independent biological replicates), and contains original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study.

Techniques: Glycoproteomics, Transfection, Western Blot, Mutagenesis, Control, Software, One-tailed Test, Immunoprecipitation, SDS Page, Immunofluorescence, Expressing, Staining, Marker

Journal: iScience

Article Title: Regulation of the endoplasmic reticulum stress sensor ATF6α through multiple oxidoreductases in the ER

doi: 10.1016/j.isci.2026.116290

Figure Lengend Snippet: Inhibition of C467 dimerization in the ATF6α transport suppressor mutant Y567N (A) Dimerization of Y567N mutant. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or Y567N mutant and analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (B) The intensity of each band, as shown in (A). The bands were quantified using the ImageJ software and normalized to the total intensity (monomer, C618 dimer, and C467 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗∗ p < 0.01). (C) C467 dimerization of Y567N and Y567N/C618A mutants. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or mutant ATF6α and analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (D) The intensity of the C467 dimer bands, as shown in (C). The bands were quantified using the ImageJ software and normalized to the total intensity (monomer, C618 dimer, and C467 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01). (E) Transport of the Y567N mutant from the ER to the Golgi apparatus. ATF6α-KO cells were transfected with p3×FLAG-tagged WT or mutant ATF6α, pretreated with 50 μM PF429242 for 1 h prior to the induction of ER stress with 5 μM Tg for 0, 30, or 60 min, and analyzed by immunoblotting with anti-FLAG antibodies under reducing or nonreducing conditions. Actin was used as a loading control. Representative immunoblots from three independent experiments are shown ( n = 3). (F) Determination of the C467 dimer level in the Golgi apparatus after 60 min of Tg treatment. The intensity of the Golgi-C467 dimer band, as shown in (E). The bands were quantified using ImageJ software and normalized to the total value (monomer, C618 dimer, C467 dimer, and Golgi-C467 dimer). The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (ns, not significant; ∗∗ p < 0.01). (G) Reduced protein levels of Y567N/C618A under ER stress. The intensity of ATF6α-F bands, as shown in (E). The bands were quantified using ImageJ, normalized to the total value (monomer, C618 dimer, C467 dimer, and Golgi-C467 dimer), and plotted against time. The means ± SDs are shown ( n = 3). Statistical analysis using a one-tailed test (∗ p < 0.05). (H and I) Interaction between ERdj5 and Y567N mutant. ATF6α-KO cells were transfected with FLAG-tagged WT or mutant ERdj5 and Myc-tagged WT or Y567N mutant ATF6α, and cell lysates were prepared for immunoprecipitation with FLAG or Myc beads. The immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies under reducing (H) and nonreducing (I) conditions. Original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study are contained in .

Article Snippet: The DOI for the Mendeley Data is listed in the . contains original uncropped western blot images used for quantification (n = 3 independent biological replicates), and contains original uncropped western blot images of at least two independent repeat experiments that support the conclusions of the study.

Techniques: Inhibition, Mutagenesis, Transfection, Western Blot, Control, Software, One-tailed Test, Immunoprecipitation, SDS Page