Ab227830

Manufactured by Abcam

Sourced in United Kingdom

Ab227830 is a recombinant human antibody that targets the protein Apolipoprotein E (ApoE). It is intended for use in research applications.

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Lab products found in correlation

9 protocols using ab227830

Redox-Sensitive Protein Regulation Protocol

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The chemical (2S, 3S)-1,4-Bis-sulfanylbutane-2,3-diol (DTT, CAS No. 3483-12-3) was obtained from Sigma; it is a strong reductant with the chemical formula C₄H₁₀O₂S₂ and MW of 154.25 g/mol. Its reducibility is largely due to the conformational stability after oxidation (containing disulfide bond). In this experiment, 3.09 g DTT powder was completely dissolved in 20 mL 0.01 M sodium acetate to obtain 1 M DTT stock solution, which was packed and stored at −20 °C before use.
Specific antibody against Nrf1 was made in our own laboratory [25 (link)]. All nine distinct antibodies against Nrf2 (ab62352), GCLC (ab207777), GCLM (ab126704), HO-1 (ab52947), GPX1 (ab108427), XBP1 (AB109221), ATF4 (ab184909), ATF6 (ab227830) and P4HB (ab137180) were obtained from Abcam (Cambridge, UK). The first three antibodies against TALDO (D623398), GSR (D220726) and NQO1 (D26104) were purchased from Sangon Biotech (Shanghai, China). The second three antibodies against BIP (bs-1219R), Chop (bs-20669R) and pIRE1 (bs-16698R) were from Bioss (Beijing, China). The third three antibodies against PSMB5 (A1975), PSMB6 (A4054), PSMB7 (A14771) were from ABclonal (Wuhan, China). Lastly, three antibodies to p-eIF2α (#5199) were from Cell Signaling Technology (Boston, MA, USA), p-PERK (sc-32577) from Santa CruZ (Santacruz, CA, USA), and β-actin (TA-09) from ZSGB-BIO (Beijing, China), respectively.

Wufuer R., Fan Z., Yuan J., Zheng Z., Hu S., Sun G, & Zhang Y. (2022). Distinct Roles of Nrf1 and Nrf2 in Monitoring the Reductive Stress Response to Dithiothreitol (DTT). Antioxidants, 11(8), 1535.

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Molecular Mechanisms of Lipid Metabolism

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DMSO, α-linolenic acid, and BSA were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). Antibodies against FASN (C2065), GAPDH (#5174), Phosoho-Akt (Ser473#4060), and Akt (#4691) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against Bax (ab32503), Bcl-2 (ab32124), IRE1 (ab124945), PERK (ab229912), and ATF6 (ab227830) were purchased from Abcam (Boston, MA, USA).

Fan H., Huang W., Guo Y., Ma X, & Yang J. (2022). α-Linolenic Acid Suppresses Proliferation and Invasion in Osteosarcoma Cells via Inhibiting Fatty Acid Synthase. Molecules, 27(9), 2741.

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Protein Extraction and Western Blotting

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Total protein was extracted from mouse tissues and cell lines by using a protein extraction buffer containing a 1% protease inhibitor cocktail (Targetmol) and a phosphatase inhibitor (Cat. No. 4906845001, Roche, Basel, Switzerland). The cell lysate was centrifuged at 4 °C at 14,000 rpm for 15 min. The obtained proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated at 4 °C overnight with the following primary antibodies: KLF4 (ab215036, Abcam, Cambridge, UK), HIF1α (66730-1-Ig, Proteintech), p-IRE1α (AP1146, ABclonal, Wuhan, China), BIP (ab108615, Abcam), ATF6 (ab227830, Abcam), Collagen I (14695-1-AP, Proteintech), Collagen III (22734-1-AP, Proteintech), GAPDH (10494-1-AP, Proteintech), and β-Tubulin (10068-1-AP, Proteintech). After washing with Tris-buffered saline with 1% Tween-20 (TBST), the membranes were incubated with HRP-conjugated secondary antibodies (ZSGB-Bio, Beijing, China) for 1 h at room temperature. The blots were visualized using an enhanced chemiluminescence reagents detection kit (Amersham Biosciences Fairfield, London, UK).

Wei S., Qi F., Wu Y, & Liu X. (2023). Overexpression of KLF4 Suppresses Pulmonary Fibrosis through the HIF-1α/Endoplasmic Reticulum Stress Signaling Pathway. International Journal of Molecular Sciences, 24(18), 14008.

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Quantification of Inflammatory Cytokines and ER Stress Markers

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The concentrations of mouse interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) were detected using enzyme linked immunosorbent assay (ELISA) kits (Elabscience, China) according to the manufacturer’s instructions. Additionally, total protein was isolated using radioimmune precipitation assay (RIPA) buffer (TaKaRa, Japan) supplemented with a protease inhibitor (Roche, Switzerland). After quantification using the BCA Protein Assay Kit (Thermo, United States), the proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated overnight with antibodies against activating transcription factor 6 (ATF6) (ab227830, Abcam, United Kingdom), p-PERK (MA5-15033, Thermo, United States), p-IRE1 (CSB-RA00,7795A724phHU, CUSABIO, China), GRP78 (MA5-27687, Thermo, United States), p-EIF2α (51995, CST, United States), CHOP (5554T, CST, United States), and GAPDH (ab8245, Abcam, United Kingdom) at 4 °C. The membranes were then incubated with HRP-conjugated secondary antibodies (Multi Sciences, China). Finally, the protein bands were detected using enhanced chemiluminescence (ECL) kits (Thermo, United States).

Chen D., Lu M.M., Wang J.H., Ren Y., Xu L.L., Cheng W.X., Wang S.S., Li X.L., Cheng X.F., Gao J.G., Kalyani F.S, & Jin X. (2022). High-fat diet aggravates colitis via mesenteric adipose tissue derived exosome metastasis-associated lung adenocarcinoma transcript 1. World Journal of Gastroenterology, 28(29), 3838-3853.

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Protein Isolation and Western Blot Analysis

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Radio immunoprecipitation assay Lysis Solution (R0010, Solarbio) was used for the isolation of proteins from cells and infarct margin tissues below the ligature line. Protein concentrations were subsequently measured with bicinchoninic acid assay protein assay kit (PC0020, Solarbio). Protein samples were separated by sodium dodecyl‐sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were sealed with 3% skimmed milk for 45 min. The primary antibodies to SPHK1 (1:500, 10670‐1‐AP, ProteinTech Group), ATF3 (1:1000, ab254268, Abcam), CHOP (1:1000, #2895, Cell Signaling Technologies), ATF6 (1:1000, ab227830, Abcam), p‐IRE1α (1:500, PA1‐16927, Thermo Fisher Scientific), IRE1α (1:500, A17940, ABclonal), p‐eIF2α (1:1000, ab32157, Abcam), eIF2α (1:500, AHO0802, Thermo Fisher Scientific), and β‐actin (1:1000, 4970, Cell Signaling Technologies) was applied overnight (4°C), followed by re‐probing with HRP‐conjugated secondary antibodies (1:4000, ab6721, Abcam or 1:5000, #31430, Thermo Fisher) for 40 min at room temperature. Finally, the protein bands were measured using ECL (32209, Thermo Fisher).

Chen H., Luo S., Chen H, & Zhang C. (2023). ATF3 regulates SPHK1 in cardiomyocyte injury via endoplasmic reticulum stress. Immunity, Inflammation and Disease, 11(9), e998.

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Protein Expression Analysis by Western Blot

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Cells were lysed using RIPA buffer containing a proteinase inhibitor (Sigma-Aldrich, USA) and phosphatase inhibitor cocktail (Bimake, USA). A Pierce BCA protein assay kit (Thermo Fisher Scientific, USA) was used to measure the protein concentration. Equal amounts of protein (30 ug) were separated by sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis, transferred onto a PVDF membrane and then detected by chemiluminescence. Antibodies used for Western blot were as follow: RUNX1 (ab23980, Abcam), RUNX1 (ab189172, Abcam), BiP (ab21685, Abcam), p-IRE1α (ab48187,S724, Abcam), ATF6 (ab227830, Abcam), XBP-1(sc-8015, Santa cruz) and the antibodies from Cell Signaling Tech were PERK(#5683), phospho-PERK(#3179), IRE1α(#3294), eIF2α(#5324), phospho-eIF2α(#3398,ser 51), ATF4(#11815), Caspase-3(#9662), GAPDH (#5174).

She C., Wu C., Guo W., Xie Y., Li S., Liu W., Xu C., Li H., Cao P., Yang Y., Wang X., Chang A., Feng Y, & Hao J. (2023). Combination of RUNX1 inhibitor and gemcitabine mitigates chemo‐resistance in pancreatic ductal adenocarcinoma by modulating BiP/PERK/eIF2α-axis-mediated endoplasmic reticulum stress. Journal of Experimental & Clinical Cancer Research : CR, 42, 238.

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Protein Quantification and Western Blot Analysis

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The cells gathered were lysed using RIPA (Beyotime, Beijing, China), and the protein concentration was measured via a bicinchoninic acid (BCA) protein quantifica-tion kit (Beyotime Biotechnology Inc., Shanghai, China). Then came SDS-polyacrylamide gel (10%) separation and transfer of proteins onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then placed at room temperature and given 5% skim milk powder blocking that lasted for 2 h, and the subsequent overnight incubation (4°C) with Ι antibodies: activating Transcription Factor 6 (ATF6; 1: 1000; ab227830; Abcam), GAPDH (1: 1000; ab9485; Abcam). Next, HRP-bound anti-rabbit IgG (ab6721, Abcam) diluted at 1: 2000 was added to incubate at an ambient temperature for 2 hours. After 3 washes with TBST buffer, the conjugated secondary antibody was tested by the enhanced chemiluminescence (ECL) system (Pierce Biotechnology, USA). GAPDH served as a control in this experiment.

Shen H., Gu J., Liu X, & Song M. (2024). Small nucleolar RNA host gene 1 silence inhibits the lipopolysaccharide-induced microglial apoptosis and inflammation. Cellular and molecular biology (Noisy-le-Grand, France), 70(3).

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Chromatin Immunoprecipitation Assay for ATF6

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ChIP assays were performed according to the manufacturer's instructions provided in the SimpleChIP Enzymatic ChIP Kit (CST). Briefly, the ChIP assay was performed using protein A/G agarose (Thermo Fisher Scientific) and an anti-ATF6 antibody (ab227830, Abcam). The immunoprecipitated DNA was used to amplify DNA fragments via PCR with specific primers. The primer sequences for GRP78 were 5′-GCGGAGCAGTGACGTTTATT-3′ (forward) and 5′-GACCTCACCGTCGCCTACT-3′ (reverse). The primer sequences for FGF2 M1 were 5′-TCTGAGCAAATAGGCCTTGCT-3′ (forward) and 5′-GGCTGAAGCCCCTGTAACAAA-3′ (reverse). The primer sequences for FGF2 M2 were 5′-ATATGCCTGGTTTTGGGCCT-3′ (forward) and 5′-CAGCCTACCGAATAGCTGGG-3′ (reverse).

Ma M., Li H., Wang P., Yang W., Mi R., Zhuang J., Jiang Y., Lu Y., Shen X., Wu Y, & Shen H. (2021). ATF6 aggravates angiogenesis-osteogenesis coupling during ankylosing spondylitis by mediating FGF2 expression in chondrocytes. iScience, 24(7), 102791.

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Western Blot Analysis of Cell Signaling Proteins

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RIPA lysis buffer was used to extract the total tissue and cellular proteins. The protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific). Equal amounts of tissue or cellular protein extracts were separated on 10% or 12% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, Eschborn, Germany). The membranes were blocked with 5% nonfat milk for 1-2 h at room temperature. Then, the blots were incubated overnight at 4 °C with primary antibodies against DAPK1 (25136-1-AP; ProteinTech), ATF6 (ab227830; Abcam), GAPDH (ab198394; Abcam), and Caspase-3 (#9662; Cell Signaling Technology). The blots were then probed with the corresponding HRP-conjugated secondary antibodies (ab150117, ab150079; Abcam) for 1-2 h at room temperature. The blots were developed using Enhanced Chemiluminescence kit (ECL; Bio-Rad Laboratories, Inc., Hercules, CA, United States). The amounts of DAPK1, ATF6 and Caspase-3 were quantified relative to GAPDH using the ChemiDoc-XRsþ (Bio-Rad).

Song Z., Li Z., Han W., Zhu C., Lou N., Li X., Luo G., Peng S., Li G., Zhao Y, & Guo Y. (2020). Low DAPK1 expression correlates with poor prognosis and sunitinib resistance in clear cell renal cell carcinoma. Aging (Albany NY), 13(2), 1842-1858.

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