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Ero1α

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ERO1α is an endoplasmic reticulum (ER) membrane protein that is involved in the oxidative protein folding process within the ER. It plays a critical role in the formation of disulfide bonds in newly synthesized proteins.

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Lab products found in correlation

S1pr1, by Abcam (2 mentions) Ptp1b, by BD (2 mentions) Cleaved caspase 3, by Cell Signaling Technology (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Ripa buffer, by Merck Group (2 mentions) β actin, by Cell Signaling Technology (2 mentions) P erk, by Cell Signaling Technology (2 mentions) Bicinchoninic acid assay kit, by Beyotime (1 mentions) Vegfa, by Abcam (1 mentions) P stat3, by Cell Signaling Technology (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions) High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)

7 protocols using ero1α

1

Immunohistochemical Analysis of Protein Expression

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Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. In citrate buffer (pH 6.0), slides were heated in an autoclave for 3 min for antigen retrieval. Slides were incubated with appropriate primary antibody (ERO1α from Santa Cruz; or S1PR1 from Abcam; or p-STAT3, VEGF-A, or CD34 from CST) overnight at 4 °C, followed by counterstaining with hematoxylin. The ERO1α expression status was graded by two independent observers as described by immunohistochemistry score: scoring was conducted based on the percentage of positive-staining cells: 0–5% scored 0, 6–35% scored 1, 36–70% scored 2, and more than 70% scored 3; staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2, and strongly staining scored 3. The final score was calculated using the percentage score × staining intensity score as follows: “0” for a score of 0–1, “1” for a score of 2–3, “2” for a score of 4–6, and “3” for a score of >6. In subsequent analyses, scores “0” and “1” were defined as the low group and scores “2” and “3” were defined as the high group scores.
Yang S., Yang C., Yu F., Ding W., Hu Y., Cheng F., Zhang F., Guan B., Wang X., Lu L, & Rao J. (2018). Endoplasmic reticulum resident oxidase ERO1-Lalpha promotes hepatocellular carcinoma metastasis and angiogenesis through the S1PR1/STAT3/VEGF-A pathway. Cell Death & Disease, 9(11), 1105.
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2

Immunoblotting of Apoptosis Markers

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Proteins extracted from aortic tissues and HAECs (10–25 μg) were separated via SDS-PAGE and transferred to Immobilon-P poly(vinylidene fluoride) membranes. Immunoblots were probed with antibodies for cleaved caspase-3 (Cell Signaling, #9661), ERO1α (Santa Cruz, sc-365526), GAPDH (Santa Cruz, sc-32233) and PTP1B (BD bioscience, #610140).
Legeay S., Fautrat P., Norman J.B., Antonova G., Kennard S., Bruder-Nascimento T., Patel V.S., Faure S, & de Chantemèle E.J. (2020). Selective deficiency in endothelial PTP1B protects from diabetes and endoplasmic reticulum stress-associated endothelial dysfunction via preventing endothelial cell apoptosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 127, 110200.
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3

Immunoblotting of Apoptosis Markers

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Proteins extracted from aortic tissues and HAECs (10–25 μg) were separated via SDS-PAGE and transferred to Immobilon-P poly(vinylidene fluoride) membranes. Immunoblots were probed with antibodies for cleaved caspase-3 (Cell Signaling, #9661), ERO1α (Santa Cruz, sc-365526), GAPDH (Santa Cruz, sc-32233) and PTP1B (BD bioscience, #610140).
Legeay S., Fautrat P., Norman J.B., Antonova G., Kennard S., Bruder-Nascimento T., Patel V.S., Faure S, & de Chantemèle E.J. (2020). Selective deficiency in endothelial PTP1B protects from diabetes and endoplasmic reticulum stress-associated endothelial dysfunction via preventing endothelial cell apoptosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 127, 110200.
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4

Quantitative Western Blot Analysis of Signaling Proteins

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Concentrations of proteins extracted from cell lines or tumor tissues were determined by bicinchoninic acid assay kits (Beyotime, Shanghai, China) following the manufacturer’s instructions. Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% skim milk powder at room temperature for 2 h, membranes were incubated with primary antibody (1:1000) overnight at 4 °C. Appropriate secondary antibody (1:2000) was added before incubation for 2 h at 37 °C. Protein band intensities were measured using Image Lab software. GAPDH was used as the internal control. Western blots were quantified using Image Pro Plus version 6. Primary antibodies were from rabbits against S1PR1, VEGF-A (Abcam, Cambridge, UK), STAT3, p-STAT3, E-cadherin, vimentin, Slug, and GAPDH (Cell Signaling Technology, Danvers, MA, USA); and from mice against ERO1α (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Yang S., Yang C., Yu F., Ding W., Hu Y., Cheng F., Zhang F., Guan B., Wang X., Lu L, & Rao J. (2018). Endoplasmic reticulum resident oxidase ERO1-Lalpha promotes hepatocellular carcinoma metastasis and angiogenesis through the S1PR1/STAT3/VEGF-A pathway. Cell Death & Disease, 9(11), 1105.
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5

Quantifying ER Stress Response Genes

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RNA was isolated with RNeasy Mini Kit (QIAGEN) and single-strand cDNA was made with High Capacity RNA-to-cDNA Kit (Applied Biosystems, Thermo Fisher Scientific). Taqman gene expression assays (Applied Biosystems, Thermo Fisher) were used to perform real-time PCR using the StepOnePlus Real-Time PCR system (Applied Biosystems, Thermo Fisher). Gene expression for PERK (Eif2ak3), ATF4 (Atf4), CHOP (Ddit3), and ERO1α (Ero1) were normalized to Gapdh. For immunoblots cell lysates were prepared in RIPA buffer (Sigma-Aldrich) and probed with PERK (Cell Signaling Technology), ERO1α (Santa Cruz Biotechnology) antibodies or β-actin (Cell Signaling Technology) loading control.
Hurst K.E., Lawrence K.A., Essman M.T., Walton Z.J., Leddy L.R, & Thaxton J.E. (2019). Endoplasmic Reticulum Stress Contributes to Mitochondrial Exhaustion of CD8+ T cells. Cancer immunology research, 7(3), 476-486.
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6

Protein Extraction and Immunoblotting for T-Cell Analysis

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T cells were lysed in RIPA Buffer (Sigma) supplemented with Protease Inhibitor Cocktail (Cell Signaling Technology) and Phosphatase Inhibitors I and II (Sigma). Protein concentrations were normalized using Pierce BCA Kit (Thermo Fisher Scientific) and loaded to 4%–10% agarose gels (Bio-Rad). P-eIF2α, eIF2α, PERK, CHOP, ATF4, β-actin, and HRP-linked anti-rabbit and mouse secondaries were obtained from Cell Signaling Technology, ERO1α was obtained from Santa Cruz Biotechnologies. Phospho protein was developed with Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific).
Riesenberg B.P., Hunt E.G., Tennant M.D., Hurst K.E., Andrews A.M., Leddy L.R., Neskey D.M., Hill E.G., Rivera G.O., Paulos C.M., Gao P, & Thaxton J.E. (2022). Stress-mediated attenuation of translation undermines T-cell activity in cancer. Cancer research, 82(23), 4386-4399.
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7

Redox State of PDI and Ero1α

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The oxidation state of PDI and Ero1α were assessed in cell lysates under both nonreducing and reducing conditions using 2-Mercaptoethanol (Calbiochem, Danvers, MA, USA) SDS-page of stain-free TGX gel (BioRad, Hercules, CA, USA) followed by immunoblotting with Ero1α (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and PDI antibodies (Cell Signaling Technology, Danvers, MA, USA).
Hurst K.E., Lawrence K.A., Reyes Angeles L., Ye Z., Zhang J., Townsend D.M., Dolloff N, & Thaxton J.E. (2019). Endoplasmic Reticulum Protein Disulfide Isomerase Shapes T Cell Efficacy for Adoptive Cellular Therapy of Tumors. Cells, 8(12), 1514.
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