p ire1α  (Novus Biologicals)

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) Schematic representation of the different IRE1 constructs used in this study. Solid red triangle (a.a. E434-D443): translocon association motif; orange dot: W457A. Each doxycycline-inducible transgene was inserted as a single copy using the Flp-In system in U-2 OS IRE1α -/- cells. (B) RT-PCR of XBP1 and GAPDH mRNAs (top), immunoblotting of IRE1α, and vinculin (VCL) (bottom) in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT and IRE1α TAD upon addition of 5 ng/mL doxycycline (Dox). Cells were treated with 5 µg/mL tunicamycin (Tm) for the indicated times. u - unspliced; s - spliced XBP1 mRNA amplicons, respectively. The asterisk indicates a hybrid amplicon generated by annealing of one strand of XBP1u and one strand of XBP1s during the PCR. GAPDH , VCL: loading controls. Quantification of the percentage of XBP1 mRNA splicing is shown in . Data are representative of three independent experiments. (C) RT-PCR and immunoblots in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α DAB and IRE1α BTAD upon addition of 5 ng/mL Dox. Cells were treated with 5 µg/mL Tm for 4 hours where indicated. Quantification of the percentage of XBP1 mRNA splicing is shown in . Data are representative of three independent experiments. (D) RT-PCR and immunoblots in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT and IRE1α DAB upon addition of 5 ng/mL doxycycline (Dox). Cells were treated with SEC61-IN-1 for 16 hours at the indicated concentrations. Quantification of the percentage of XBP1 mRNA splicing is shown in . Data are representative of three independent experiments. (E) Immunoprecipitation of mNeonGreen-tagged IRE1α WT in U-2 OS IRE1α -/- cells expressing IRE1α WT upon addition of 5 ng/mL Dox. Cells were treated with 5 µg/mL Tm for 6 hours or 1 µM SEC61-IN-1 for 6 and 16 hours as indicated. Immunoblotting was performed using antibodies against SEC61α and IRE1α. Lanes are cropped from images of the same gel. Data are representative of two independent experiments. (F) Immunoblots of phosphorylated and total IRE1α (top) and spliced XBP1 protein and vinculin (bottom) in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox. Cells were treated with CADA for 16 hours at the indicated concentrations. The quantification of the proportion of phosphorylated IRE1 and spliced XBP1 are shown below the immunoblots. Data are representative of two independent experiments. VCL: loading control. (G) RT-PCR of XBP1 mRNA in H4 cells depleted of SRP54 by RNA interference. Quantification of the percentage of XBP1 mRNA splicing is shown in . Data are representative of three independent experiments. Bottom panel: extent of SRP54 knockdown measured by RT-qPCR and normalized internally to GAPDH . Mean ± SEM expressed as fold change over the levels in mock-transfected controls (N=3).

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Amplification, Generated, Immunoprecipitation, Control, Knockdown, Quantitative RT-PCR, Transfection

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) Immunoprecipitation of mNeonGreen-tagged IRE1α WT and IRE1α TAD in lysates obtained from U-2 OS IRE1α -/- cells expressing the respective constructs after the addition of 5 ng/mL Dox for 48 hours. Immunoblotting was performed using antibodies against SEC61α and IRE1α. (B-C) RT-PCR of XBP1 and GAPDH mRNAs in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT and IRE1α DAB (B) or mNeonGreen-tagged IRE1 BTAD (C) upon addition of 5 ng/mL doxycycline (Dox). Cells were treated with 5 µg/mL Tm for 4 hours, 500 µM palmitic acid for 6 hours, or 1 µM CVT-11127 for 24 hours as indicated. u - unspliced; s - spliced XBP1 mRNA amplicons, respectively. The asterisk indicates a hybrid amplicon generated by annealing of one strand of XBP1 u and one strand of XBP1 s during the PCR. GAPDH: loading control. Data are representative of two independent experiments. (D) Immunoblots of IRE1α and tubulin in lysates obtained from U-2 OS ( IRE1α +/+ ) cells and U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT , IRE1α TAD , IRE1α DAB , and IRE1α BTAD upon addition of 5 ng/mL doxycycline (Dox) for 48 hours (left). Relative protein expression levels of IRE1 were normalized to tubulin and quantified as a fold change of the mean ± SEM over endogenous IRE1α levels (right) (N=3). (E) RT-qPCR of IRE1α mRNA levels in lysates obtained from U-2 OS ( IRE1α +/+ ) cells and U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT , IRE1α TAD , IRE1α DAB , and IRE1α BTAD upon addition of 5 ng/mL doxycycline (Dox) for 48 hours. Relative IRE1α mRNA expression levels were normalized internally to the reference gene GAPDH and expressed as log 2 (fold change) over the levels in U-2 OS ( IRE1α +/+ ) cells. Data: mean ± SEM (N=6). (F-G) Quantification of XBP1 mRNA splicing from experiments shown in (F) and (G). Data: mean ± SD (N ≥ 2).

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Immunoprecipitation, Expressing, Construct, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Generated, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) Quantification of XBP1 mRNA splicing from experiments shown in . Data: mean ± SD (N ≥ 2). (B) Molecular docking of SEC61-IN-1 into the structure of the SEC61 translocon, and Cryo-EM structure of CADA bound to the SEC61 translocon (PDB: 8DO2). Panels on the right show the zoomed in views of the small molecules occupying the central pore of SEC61α. (C) Immunoblots of XBP1s, Vinculin (VCL) and IRE1α in lysates obtained from U-2 OS cells treated with CADA and SEC61-IN-1 for 16 hours at the indicated concentrations. The quantification of the proportion of spliced XBP1 protein is indicated below. Immunoblots for each treatment were processed in parallel on different gels. Data are representative of two independent experiments. (D) Quantification of XBP1 mRNA splicing from experiments shown in . Data: mean ± SD (N ≥ 2).

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Cryo-EM Sample Prep, Western Blot

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: Representative micrographs of U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL doxycycline and treated with 1 µM SEC61-IN-1 or 5 µg/mL Tm for the indicated times. Arrowheads indicate high-order mNeonGreen-tagged IRE1α foci observable by confocal fluorescence microscopy. Scale bar = 10 µm.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Expressing, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) Quantification of ribosome density based on analysis of transmission electron micrographs of ER membranes (shaded in purple) in U-2 OS cells treated with 100 nM RocA for 16 hours. Electron dense ribosome particles are highlighted in blue. Data: Mean ± SEM (N=22 micrographs for control and N=17 micrographs for RocA). (B-D) Quantification of XBP1 mRNA splicing from experiments shown in , 2D and 2E. Data: mean ± SD (N ≥ 2). (E) Immunoblots of XBP1s, IRE1α, and Vinculin (VCL) in lysates obtained from U-2 OS cells treated with RocA for 16 hours at the indicated concentrations or 5 µg/mL Tm for 4 hours. The quantification of the proportion of spliced XBP1 protein is indicated below the immunoblots. Data are representative of three independent experiments. (F) RT-qPCR of SCARA3 and COL6A1 mRNAs in H4 cells treated with 1 µM RocA for 16 hours. Expression levels were normalized internally to 28S ribosomal RNA. Values are expressed as fold change over the levels in H4 cells treated with DMSO (indicated with a dashed line). Data: Mean ± SEM (N = 3). (G) U-2 OS cells treated with RocA at the indicated concentrations for 8 hours were subjected to puromycin pulse-chase analyses to assess nascent protein synthesis rates. Immunoblotting of lysates was performed using antibodies to puromycin and VCL. Data are representative of two independent experiments. Lanes were cropped from the same gel. (H) Confocal micrographs of U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox and treated with 100 nM RocA for the indicated times. Data are representative of two independent experiments. Scale bar = 10 µm.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Transmission Assay, Control, Western Blot, Quantitative RT-PCR, Expressing, Pulse Chase

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) Immunoprecipitation of mNeonGreen-tagged IRE1α WT in U-2 OS IRE1 α -/- cells expressing IRE1α WT upon addition of 5 ng/mL Dox. Cells were treated with 500 nM RocA for 16 hours as indicated. Immunoblotting was performed using antibodies against SEC61α, RPL9, and IRE1α. Data are representative of two independent experiments. (B) RT-PCR of XBP1 and GAPDH mRNAs (top), immunoblotting of IRE1α, and VCL (bottom) in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox. Cells were treated with RocA for 16 hours at the indicated concentrations or 5 µg/mL Tm for 4 hours. u - unspliced; s - spliced XBP1 mRNA amplicons, respectively. The asterisk indicates a hybrid amplicon generated by annealing of one strand of XBP1u and one strand of XBP1s during the PCR. GAPDH , VCL: loading controls. Quantification of the percentage of XBP1 mRNA splicing is shown in . Data are representative of three independent experiments. (C) Immunoprecipitation of mNeonGreen-tagged IRE1α DAB as in panel A. (D-E) RT-PCR and immunoblotting as in panel B of lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α DAB (D), and mNeonGreen-tagged IRE1α TAD (E). Quantification of the percentage of XBP1 mRNA splicing is shown in . Data are representative of three independent experiments. (F) RT-PCR of XBP1 and GAPDH mRNAs in lysates obtained from U-2 OS IRE1α +/+ cells and U-2 OS IRE1α -/- cells expressing IRE1α WT cells upon addition of 5 ng/mL Dox. Cells were treated with 100 nM RocA for 16 hours or 5 µg/mL Tm for 4 hours as indicated. Data are representative of three independent experiments. (G) Immunoblots of phosphorylated and total IRE1α (top) and spliced XBP1 protein and VCL (bottom) in lysates obtained from U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox. Cells were treated with RocA for 16 hours at the indicated concentrations or 5 µg/mL Tm for 4 hours as indicated. The quantification of the proportion of phosphorylated IRE1 and spliced XBP1 are shown below the immunoblots. Data are representative of three independent experiments. GAPDH: loading control.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Immunoprecipitation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Generated, Control

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) Schematic representation of FKBP-PKR. The homodimerizer is shown in red. The FKBP F36V domain (light blue) is adjoined to the PKR kinase domain (dark blue) by PKR’s unstructured linker. (B) Co-immunoprecipitation of IRE1α and SEC61α in lysates obtained from H4 cells expressing FKBP-PKR and treated with the homodimerizer for 16 hours. (C) Immunoblot of IRE1α phosphorylation (IRE1α-P) and RT-PCR of XBP1 mRNA and 28S ribosomal RNA in lysates obtained from H4 cells expressing FKBP-PKR and treated with the homodimerizer for the indicated times. Loading control: 28S ribosomal RNA. Data are representative of five independent experiments. (D) RT-qPCR of BLOC1S1 and SCARA3 mRNAs in H4 cells expressing FKBP-PKR and treated with the homodimerizer for 16 hours with or without co-treatment with 10 µM of the IRE1 RNase inhibitor 4µ8C. Data: Mean ± SEM of fold changes normalized to the levels in untreated controls (N≥3). (E) Immunoblot of IRE1α phosphorylation (IRE1α-P) and RT-PCR of XBP1 and GFP mRNAs in lysates obtained from H4 cells expressing eIF2α S51D hosted in a construct that also expresses GFP, for the indicated times. GFP mRNA: transfection and loading control. Data are representative of three independent experiments. The ♢ indicates a non-specific band in the immunoblot (top) and * denotes the hybrid XBP1 amplicon in the RT-PCR (bottom). (F) RT-qPCR of BLOC1S1 and SCARA3 mRNAs in H4 cells expressing eIF2α S51D for 16 hours. Data: Mean ± SEM of fold changes normalized to the levels in untreated controls (N = 3). (G) RT-PCR of XBP1 mRNA and 28S ribosomal RNA in H4 cells expressing FKBP-PKR and treated with the homodimerizer for 16 hours with or without co-treatment with 800 nM ISRIB. Data are representative of three independent experiments. (H) RT-qPCR of the expression levels of the BLOC1S1 mRNA in H4 cells expressing FKBP-PKR, treated with the homodimerizer for 16 hours with or without addition of 800 nM ISRIB. Data: Mean ± SEM of fold changes normalized to the respective levels in untreated controls (N = 3). **p-value < 0.01, Student’s t-test. (I) Immunoblots of PERK and ATF4 in lysates obtained from H4 cells expressing FKBP-PKR and treated with the homodimerizer for the indicated times and 5 µg/ml Tm for 4 hours. Actin: loading control. (J) left panel: RT-qPCR of the ATF6 mRNA in H4 cells with CRISPRi-mediated knockdown of ATF6 (sgATF6); right panel: HSPA5 mRNA in H4 cells expressing FKBP-PKR cells under the following conditions: 2.5 µg/ml Tm for 16 hours, 5 nM Ceapin-A7, and homodimerizer treatment for 16 hours, as indicated, or in cells with CRISPRi-mediated knockdown of ATF6 (sgATF6). For the right panel data: Mean ± SEM of fold changes normalized to the levels in untreated controls. **p-value <0.01, Student’s t-test; n.s. - not significant. (K) RT-PCR of XBP1 and GAPDH mRNAs in U-2 OS IRE1α -/- cells expressing FKBP-PKR and mNeonGreen-tagged IRE1α DAB treated with 20 ng/mL Dox for 48 hours, 5 µg/mL Tm for 4 hours as indicated, and the homodimerizer for the indicated times. Data are representative of three independent experiments. The asterisk indicates the hybrid XBP1 amplicon in the RT-PCR.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Immunoprecipitation, Expressing, Western Blot, Phospho-proteomics, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative RT-PCR, Construct, Transfection, Amplification, Knockdown

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A) H4 cells expressing FKBP-PKR and treated with the homodimerizer for the indicated times were subjected to puromycin pulse-chase analyses to assess nascent protein synthesis rates. Immunoblotting was performed using antibodies to puromycin. Ponceau staining: loading control. Data are representative of three independent experiments. (B) RT-PCR of XBP1 and GAPDH mRNAs in U-2 OS IRE1α -/- cells expressing FKBP-PKR and mNeonGreen-tagged IRE1α WT . IRE1α WT expression was induced with 20 ng/mL doxycycline (Dox). Cells were treated with 5 µg/mL Tm for 4 hours or the homodimerizer for the indicated times. Data are representative of three independent experiments. (C-D) RT-qPCR of DNAJB9 (C) and HSPA5 (D) mRNAs in H4 cells expressing FKBP-PKR and treated with the homodimerizer. Data: Mean ± SEM of fold changes normalized to the levels in untreated controls; N = 4 for (C), and N = 5 for (D). (E) Immunoblots of FLAG-tagged eIF2α S51D , ATF4, GADD34, and GFP in lysates obtained from H4 cells expressing eIF2α S51D hosted in a construct that also expresses GFP for 24 hours. GFP: loading control. (F-G) RT-qPCR of DNAJB9 (F) and HSPA5 (G) mRNAs in H4 cells expressing eIF2α S51D hosted in a construct that also expresses GFP for the indicated times. Data: Mean ± SEM of fold changes normalized to the levels in untreated controls (N ≥ 2). (H-I) RT-qPCR of DNAJB9 (H) and HSPA5 (I) mRNAs in H4 cells expressing FKBP-PKR and treated with the dimerizer, with or without co-treatment with 800 nM ISRIB, for 16 hours. Data: Mean ± SEM of fold changes normalized to the levels in untreated controls (N = 3). *p-value < 0.05, **p-value < 0.01, Student’s t-test. (J) RT-PCR of XBP1 mRNA in H4 cells treated with 10 µM BtdCPU with or without co-treatment with 800 nM ISRIB for 24 hours. Data are representative of three independent experiments. (K) RT-PCR of XBP1 mRNA and 28S ribosomal RNA in HEK-293 cells treated with 5 ng/µl (6.3 µM) oligomycin (OMY) with or without co-treatment with either 10 µM 4µ8C or 800 nM ISRIB for 16 hours. Data are representative of three independent experiments. (L) RT-qPCR of GRP94 mRNA in H4 cells expressing FKBP-PKR and treated with the homodimerizer for 16 hours. Data: Mean ± SEM of fold changes normalized to the levels in control cells. (M) Confocal micrographs of U-2 OS IRE1α -/- cells expressing FKBP-PKR and mNeonGreen-tagged IRE1α WT and treated with 300 nM thapsigargin (Tg) for 4 hours or the homodimerizer for 16 hours (N = 3). Scale bar = 10µm.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Expressing, Pulse Chase, Western Blot, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Construct

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A-B) RNA-sequencing based validation of XBP1 mRNA splicing in (A) U-2 OS IRE1α -/- cells, U-2 OS IRE1α +/+ cells, and U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox and treated with 100 nM RocA for 16 hours or Tm for 4 hours as indicated; and (B) H4 cells expressing FKBP-PKR and treated with the homodimerizer for the indicated times. Data: Proportion of XBP1s (Ratio of counts per million XBP1 reads).

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: RNA Sequencing, Biomarker Discovery, Expressing

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A-B) Bubble plots showing enriched Reactome pathways to which IRE1-dependent transcripts cluster as upregulated in (A) U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1ɑ WT upon addition of 5 ng/mL Dox and treated with 100 nM RocA for 16 hours (log 2 FoldChange > 0.5 compared to DMSO controls), and (B) H4 cells with the activation of FKBP-PKR for 16 hours and 24 hours (continuous enrichment test as compared to controls). (C-D) Heatmaps showing log 2 FoldChanges of specific transcripts in U-2 OS IRE1α -/- cells, U-2 OS IRE1α +/+ cells, and U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1ɑ WT upon addition of 5 ng/mL Dox. Cells were treated with 100 nM RocA for 16 hours as indicated. In parallel, U-2 OS IRE1α -/- cells and U-2 OS IRE1α +/+ cells were treated with Tm for 4 hours. All log 2 FoldChanges were calculated in comparison to DMSO controls. The Reactome pathways to which the protein products of the differentially expressed transcripts belong are highlighted on the left of each panel.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Comparison

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: (A-B) Bubble plots showing differentially enriched Reactome pathways to which IRE1-dependent transcripts cluster as upregulated in (A) U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox and treated with 100 nM RocA for 16 hours (log 2 FoldChange > 0.5 compared to DMSO controls), and (B) H4 cells with the activation of FKBP-PKR for 16 hours and 24 hours (continuous enrichment test as compared to controls). (C) Heatmap showing log 2 FoldChanges of common UPR transcripts in U-2 OS IRE1α -/- cells, U-2 OS IRE1α +/+ cells, and U-2 OS IRE1α -/- cells expressing mNeonGreen-tagged IRE1α WT upon addition of 5 ng/mL Dox. Cells were treated with 100 nM RocA for 16 hours. In parallel, U-2 OS IRE1α -/- and U-2 OS IRE1α +/+ cells were treated with Tm for 4 hours. All log 2 FoldChanges are relative to DMSO controls.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Expressing, Activation Assay

Journal: bioRxiv

Article Title: IRE1 drives a homeostatic response to reduced protein influx into the endoplasmic reticulum

doi: 10.64898/2026.03.02.709157

Figure Lengend Snippet: Schematic model: (1) Co-translational translocation of signal-peptide bearing proteins into the ER facilitates IRE1’s association with the SEC61 translocon. These IRE1 species likely co-exist in a dynamic equilibrium. (2) The IRE1-SEC61 complex functions as an internal limiter of IRE1 activity when the ER protein load is optimal (the ‘Goldilocks zone’ (3) of ER protein load). (4) During TRES, disrupted co-translational translocation and reduced ER protein influx result in the closure of the SEC61 channel and the subsequent eviction of IRE1 from the translocon complex, leading to IRE1’s derepression and TRES signaling. (5) IRE1-driven gene expression programs in-turn prepare the cell for increased rates of protein synthesis and translocation into the ER by upregulating components of the co-translational translocation machinery. Inset: U-shaped response of IRE1 activity as a function of ER protein load.

Article Snippet: 500 μg of supernatant was then incubated overnight, rotating at 4 °C, with 2.5 μg of the anti-IRE1α antibody (Cell Signaling Technology).

Techniques: Translocation Assay, Activity Assay, Gene Expression