Rabbit anti p eif2α

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-p-eIF2α is a primary antibody that detects phosphorylated eIF2α (Ser51). eIF2α is a key regulator of protein synthesis and its phosphorylation is an important indicator of the cellular stress response.

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Lab products found in correlation

11 protocols using rabbit anti p eif2α

Comprehensive Antibody Panel for Autophagy and UPR Analysis

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Rabbit anti-LC3B (#3868), rabbit anti-ATG5 (#12994), rabbit anti-ATG7 (#8558), rabbit anti-Beclin-1 (#3495), rabbit anti-ATG16L1 (#8089), rabbit anti-BIP (#3177), rabbit anti-ATF6 (#65880), rabbit anti-ATF4(#11815), rabbit anti-XBP1 (#12782), rabbit anti-PERK (#5683), rabbit anti-IRE1 (#3294), rabbit anti-p-eif2α (#3398), rabbit anti-FIP200 (#12436), and mouse anti-CHOP (#2895) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) (dilution concentration 1:1000). Rabbit anti-p-IRE1 (PA1-16927) was purchased from Invitrogen (Waltham, MA, USA) (dilution concentration 1:1000). Mouse anti-p62 (18420-1-AP), mouse anti-HA-tag (66006-2-Ig), and mouse anti-β-actin (66009-1-Ig) were purchased from Proteintech (Wuhan, China) (dilution concentration 1:5000). HRP-conjugated goat anti-mouse (G1214) and goat anti-rabbit (G1213) were purchased from Servicebio (Wuhan City, China) (dilution concentration 2:5000). Alexa Fluor 568 goat anti-mouse IgG, IgM (H + L) (A-11004) and Alexa Fluor 647 goat anti-mouse-IgG (H + L) (A-21445) were purchased from Thermo Fisher (Waltham, MA, USA) (dilution concentration 1:500). Immunofluorescence antibodies were diluted in PBS. Immunoblot antibodies were diluted in TBST.

Su W.Q., Yu X.J, & Zhou C.M. (2021). SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response. Viruses, 13(12), 2467.

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Western Blot Analysis of Hepatic Signaling

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Hepatic tissues and cells prepared with radioimmunoprecipitation (RIPA) buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1 mM NaF, 1% NP40 and 0.1% SDS, supplemented with protease and phosphatase inhibitors (Millipore, USA). 50 μg lysates were loaded onto 10% SDS-PAGE and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, USA). The proteins were visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) according to the manufacturer’s protocol. Western blots were performed using antibodies against rabbit anti-p-AKT (Cell Signaling, #4060, 1:1000), rabbit anti-t-AKT (Cell Signaling, #9272, 1:2000), goat anti-TRB3 (Santa Cruz, sc-34211, 1:500), mouse anti-CHOP (Beyotime, #AC532, 1:1000), rabbit anti-BIP (Beyotime, #AB310, 1:1000), rabbit anti-p-eIF2α (Cell Signaling, #9721, 1:1000), rabbit anti-t-eIF2α (Cell Signaling, #9722, 1:1000), rabbit anti-p-IRE1α (Novus Biologicals, #NB100-2323, 1:1000), rabbit anti-t-IRE1α (Cell Signaling, #3294, 1:1000) and rabbit anti-GAPDH (Cell Signaling, #5174, 1:1000).

Liu X., Zhao J., Liu Q., Xiong X., Zhang Z., Jiao Y., Li X., Liu B., Li Y, & Lu Y. (2016). MicroRNA-124 promotes hepatic triglyceride accumulation through targeting tribbles homolog 3. Scientific Reports, 6, 37170.

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Western Blotting for Protein Expression

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Western blotting was performed using established methodology [6 (link),28 (link)]. Briefly, cells were washed with PBS and lyzed in RIPA buffer. Proteins (20–30 μg) were separated by SDS/PAGE (12% gel) and then transferred on to an immunoblot PVDF membrane (Bio-Rad, Hercules, CA). After blocking in PBS/Tween (0.1%) with 5% non-fat milk, the membrane was incubated with primary antibodies overnight at 4°C followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, 1:3000) and then developed using ECL solution (Pierce). Primary antibodies used were mouse anti-APOL1 (Proteintech, 66124-1-lg, 1:1000), rabbit anti-nephrin (Abcam, ab58968, 1:1000), rabbit anti-podocin (Sigma, P0372, 1:2000), rabbit anti-GRP78 binding Ig protein (BiP) (Abcam, ab21685, 1:1000), rabbit anti-p-eIF2α (Cell Signaling Technology, #9721, 1:1000), and mouse anti-actin (Santa Cruz Biotechnology, sc-8432, 1:3000). For protein expression quantitation, the films were scanned with a CanonScan 9950F scanner and the acquired images were then analyzed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image/).

Wen H., Kumar V., Lan X., Shoshtari S.S., Eng J.M., Zhou X., Wang F., Wang H., Skorecki K., Xing G., Wu G., Luo H., Malhotra A, & Singhal P.C. (2018). APOL1 risk variants cause podocytes injury through enhancing endoplasmic reticulum stress. Bioscience Reports, 38(4), BSR20171713.

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Western Blot Analysis of Protein Expression

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Cells were lysed with Laemmli's Blue. After boiling for 5 min, lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel and proteins were transferred to a nitrocellulose membrane. After blocking with TBS 5% bovine serum albumin, the membrane was incubated with rabbit anti-pIgR (1:6000; homemade Lp877), rabbit anti-GAPDH (1:3000; Sigma-Aldrich), mouse anti-eIF2α (1:2000; Cell Signaling), rabbit anti-P-eIF2α (1:1000; Cell Signaling), and mouse anti-β-actin (1:1000; Sigma-Aldrich) overnight at 4°C, then with goat anti-rabbit IgG (1:2000; Cell Signaling) or anti-mouse IgG (1:5000; Sigma-Aldrich) for 1 h at room temperature. Detection of immunoreactivity was performed with the ECL^TM Prime Western Blotting Detection chemiluminescence reagent (Amersham^TM ECL, GE Healthcare, UK) blot and quantification was performed by using Quantity One software.

Collin A.M., Lecocq M., Noel S., Detry B., Carlier F.M., Aboubakar Nana F., Bouzin C., Leal T., Vermeersch M., De Rose V., Regard L., Martin C., Burgel P.R., Hoton D., Verleden S., Froidure A., Pilette C, & Gohy S. (2020). Lung immunoglobulin A immunity dysregulation in cystic fibrosis. EBioMedicine, 60, 102974.

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Endoplasmic Reticulum Stress Response Evaluation

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Poly-D-lysine hydrobromide, amprolium, and laminin were purchased from Sigma (St. Louis, MO, USA). Rabbit anti-p-eIF2α, anti-caspase-12, anti-ATF6, anti-dinitrophenol (DNP) antibodies were obtained from Cell signaling Technology (Danvers, MA). Rabbit anti-GRP78 antibody was obtained from Novus Biologicals (Littleton, CO). Mouse anti-CHOP antibody was obtained from Thermo Fisher Scientific (Rockford, IL). Rabbit anti-4-hydroxynonenal (HNE) antibody was obtained from lifespan BioSciences (Seattle, WA). HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from GE Healthcare life Sciences (Piscataway, NJ). Calcein AM was obtained from Thermo Fisher Scientific (Waltham, MA).

Wang X., Xu M., Frank J.A., Ke Z.J, & Luo J. (2017). Thiamine deficiency induces endoplasmic reticulum stress and oxidative stress in human neurons derived from induced pluripotent stem cells. Toxicology and applied pharmacology, 320, 26-31.

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Immunoblotting and Immunohistochemistry of ER Stress Markers

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Tunicamycin and mouse anti-glial fibrillary acidic protein (GFAP) antibody were obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit anti-ATF6 antibody was purchased from LifeSpan Biosciences (Seattle, WA). Rabbit anti-Xbp1s antibody was purchased from Biolegend (San Diego, CA). Rabbit anti-p-eIF2α and cleavedcaspase-3 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Rabbit anti-GRP78 antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas). Rat anti-GRP94 antibody was obtained from Enzo Life Sciences (Farmingdale, NY 11735). Rabbit anti-MANF antibody was purchased from Abcam (Cambridge, MA). Mouse antineuronal nuclei (NeuN) antibody was obtained from Millipore Corporate (Billerica, MA). Mouse anti-tubulin, HRP-conjugated anti-rabbit, anti-mouse and anti-rat secondary antibodies were purchased from GE Healthcare Life Sciences (Piscataway, NJ). Biotin-conjugated anti-rabbit secondary antibodies and ABC kit were obtained from Vector Laboratories (Burlingame, CA). Alexa-488 conjugated anti-rabbit and Alexa-594 conjugated anti-mouse antibodies were obtained from Life Technologies (Grand Island, NY). Ketamine/xylazine was obtained from Butler Schein Animal Health (Dublin, OH). Other chemicals and reagents were purchased either from Sigma Chemical or Life Technologies.

Wang H., Wang X., Ke Z.J., Comer A.L., Xu M., Frank J.A., Zhang Z., Shi X, & Luo J. (2015). Tunicamycin-induced Unfolded Protein Response in the Developing Mouse Brain. Toxicology and applied pharmacology, 283(3), 157-167.

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Measuring Protein Synthesis and Signaling

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Whole-cell extracts from cell lines and islet lysates were prepared and subjected to immunoblot analysis as described previously^{11 (link)}. For immunoblotting of puromycin incorporation into protein, puromycin was added to the culture medium at 1 ug/mL during the final 15 minutes of incubation. Cells were then washed twice with cold PBS and lysed. Immunoblot analyses were performed after separation of protein extracts on a 4% to 20% gradient SDS-polyacrylamide gel and were visualized using fluorescently labeled primary antibody to puromycin (mouse anti-puromycin, 1:1000 concentration, Kerafast, Catalog number EQ0001), p-4EBP1 (rabbit anti-p-4EBP1, 1:1000, Cell Signaling Technology, number 9455), p-p70 S6K (Thr389) (rabbit anti-p-p70S6K, 1:1000, Cell Signaling Technology, number 2708), p-eIF2α (rabbit anti-p-eIF2 α, 1:1000, Cell Signaling Technology, number 9721), and secondary antibodies (Li-Cor Biosciences) and quantified using a Li-Cor Odyssey scanner and Image J 1.38x^{50 (link)}.

Hatanaka M., Anderson-Baucum E., Lakhter A., Kono T., Maier B., Tersey S.A., Tanizawa Y., Evans-Molina C., Mirmira R.G, & Sims E.K. (2017). Chronic high fat feeding restricts islet mRNA translation initiation independently of ER stress via DNA damage and p53 activation. Scientific Reports, 7, 3758.

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Immunoblot and Immunofluorescence Antibody Protocols

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The following antibodies were used for immunoblot analysis: rabbit-anti-IKK2 (CST-2678, Cell Signaling, Danvers, MA, USA), rabbit-anti-p-p65 (CST-3033, Cell Signaling, Danvers, MA, USA), rabbit-anti-p65 (sc-372, Dallas, TX, USA), chicken-anti-GFP (ab13970, Cambridge, UK), rabbit-anti-tubulin (ab6046, Cambridge, UK), rabbit-anti-PLP1 (CST-28702, Cell Signaling, Danvers, MA, USA), rabbit-anti-MOG (CST-96457, Cell Signaling, Danvers, MA, USA), rabbit-anti-MBP (CST-2396, Cell Signaling, Danvers, MA, USA), rabbit-anti-GAPDH (CST-3686, Cell Signaling, Danvers, MA, USA), rabbit-anti-p-eIF2α (CST-3597, Cell Signaling, Danvers, MA, USA), rabbit-anti-eIF2α (CST-5324, Cell Signaling, Danvers, MA, USA), rabbit-anti-ERK2 (sc-1647, Dallas, TX, USA).
For immunofluorescence, the following primary antibodies were used: mouse-anti-CC1 (OP80, Merck, Darmstadt, Germany), mouse-anti-GSTπ (BD610719, BD, Franklin Lakes, NJ, USA), chicken-anti-GFP (GFP-1020, AvesLab, Davis, CA, USA), mouse-anti-GFAP (sc-33673, Dallas, TX, USA), rabbit-anti-NG2 (AB5320, Merck, Darmstadt, Germany), rabbit-anti-MBP (Biolegend 836,504, San Diego, CA, USA), mouse-anti-Neurofilament-H (SMI32P) (Biolegend 801,701, San Diego, CA, USA), rabbit-anti-ß3-tubulin (Biolegend 802,001, San Diego, CA, USA), rabbit- anti RFP (abcam ab124754, Cambridge, UK).

Schlett J.S., Mettang M., Skaf A., Schweizer P., Errerd A., Mulugeta E.A., Hein T.M., Tsesmelis K., Tsesmelis M., Büttner U.F., Wendt H., Abaei A., Rasche V., Prex V., Nespoli E., Alami N.O., Tews D., Walther P., Yilmazer-Hanke D., Oswald F., Dimou L., Wirth T, & Baumann B. (2023). NF-κB is a critical mediator of post-mitotic senescence in oligodendrocytes and subsequent white matter loss. Molecular Neurodegeneration, 18, 24.

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Immunohistochemical Staining of Larval Wing Discs

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Larval wing imaginal discs were stained using the following antibodies: Rabbit anti-cleaved Dcp-1 (1:300, Cell Signaling Technology), Rabbit-anti-p-eIF2α (1:300, Cell Signaling Technology), Mouse-anti-dMyc (1:20, a gift from Bruce Edgar lab, University of Utah, USA), Mouse-anti-beta Galactosidase (1:50, 40-1A, DSHB) and Hoechst 33342, H3570 (1:1000, Invitrogen). Fluorescent secondary antibodies used were Alexa-405, Alexa-488, Alexa-594, and Alexa-647 (Invitrogen) at 1:500 dilutions.

Paul P.K., Umarvaish S., Bajaj S., S. R.F., Mohan H., Annaert W, & Chaudhary V. (2024). Maintenance of proteostasis by Drosophila Rer1 is essential for competitive cell survival and Myc-driven overgrowth. PLOS Genetics, 20(2), e1011171.

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Western Blot Analysis of Protein Extracts

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Protein extracts (100 μg) were separated by sodium dodecyl sulfate polyacrylamide (40% acrylamide/bis 19:1, BioRad) gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). For immunoblots, the antibodies used at a 1/1000 dilution were rabbit anti-eIF2α (Santa Cruz), rabbit anti-eIF2α-P (Cell Signaling), mouse anti-cdc2 (Abcam), mouse anti-actin (MP Biomedicals) or mouse anti-GFP (Roche), followed by anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (Promega), used at a 1/5000 dilution. After extensive washing, the immunoreactive bands were detected using the Immobilon Crescendo Western HRP substrate (Millipore).

Jiménez-Saucedo T., Berlanga J.J, & Rodríguez-Gabriel M. (2021). Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan. Aging (Albany NY), 13(8), 10989-11009.

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