P eif2a

Manufactured by Cell Signaling Technology

Sourced in United States

P-eIF2a is an antibody that recognizes the phosphorylated form of the eukaryotic translation initiation factor 2 alpha (eIF2α) protein. The phosphorylation of eIF2α is a key regulatory mechanism in the control of protein synthesis.

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Lab products found in correlation

26 protocols using p eif2a

CYT997 Protocol for Cellular Assays

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CYT997 (MF: C₂₄H₃₀N₆O₂, MW: 434.53, purity: 99.46%) was purchased from Selleckchem (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO) to prepare a 40 mM stock solution, which was stored at − 80 °C. DMSO was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), N-acetylcysteine (NAC), 3-methyladenine (3-MA), chloroquine (CQ) and GSK2606414 were purchased from Sigma-Aldrich. EN460 was purchased from MedchemExpress (Monmouth Junction, NJ, USA). Antibodies against PARP, C-PARP, CASPASE-4, LC3B-I/II, BECLIN-1, PERK, P-PERK, EIF2A, P-EIF2A, CHOP, ERO1-Lα, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).

Wang Z., Yin F., Xu J., Zhang T., Wang G., Mao M., Wang Z., Sun W., Han J., Yang M., Jiang Y., Hua Y, & Cai Z. (2019). CYT997(Lexibulin) induces apoptosis and autophagy through the activation of mutually reinforced ER stress and ROS in osteosarcoma. Journal of Experimental & Clinical Cancer Research : CR, 38, 44.

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Immunoblotting Protocol for ER Stress Proteins

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Cell lysis was performed in RIPA buffer (Boston Bio Products) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). SDS-PAGE was performed on NuPage Bis-Tris gels (ThermoFisher Scientific) using MOPS or MES running buffer. Gels were dry transferred onto 0.45 μm nitrocellulose membranes using the iBlot® Dry Blotting System (ThermoFisher Scientific).
The following Abs were purchased from Cell Signaling Technology: EIF2A (#5324), p-EIF2A (#3398), ATF4 (#11815), CHOP (2895), CALR (#12238), cGAS (#15102), TBK1 (#3504), pTBK1 (#5483), pIRF3 (#29047), STING (#13647). GAPDH (#2118) and B-ACTIN (#4970) were used as loading controls.

Gulla A., Morelli E., Samur M.K., Botta C., Hideshima T., Bianchi G., Fulciniti M., Malvestiti S., Prabhala R.H., Talluri S., Wen K., Tai Y.T., Richardson P.G., Chauhan D., Sewastianik T., Carrasco R.D., Munshi N.C, & Anderson K.C. (2021). Bortezomib Induces Anti–Multiple Myeloma Immune Response Mediated by cGAS/STING Pathway Activation. Blood Cancer Discovery, 2(5), 468-483.

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Endoplasmic Reticulum Stress Pathway Markers

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The ATF4/6-inducible reporter plasmid (pATF4/6-Luc) was purchased from Addgene (Watertown, MA, USA). Rabbit polyclonal antibodies against GRP78, PERK, p-eIF2a, eIF2a and IRE1 were supplied by Cell Signaling Technology (Danvers, MA, USA). The rabbit monoclonal antibody against cleaved casepase-3 was purchased from Cell Signaling Technology (Danvers, MA, USA). The mouse monoclonal antibody against CHOP was supplied by Cell Signaling Technology (Danvers, MA, USA). The rabbit polyclonal antibody against p-IRE1 was provided by Abcam (Cambridge, England). The rabbit polyclonal antibody against ATF6 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse monoclonal antibodies against Flag and β-actin were obtained from Sigma (St Louis, MO, USA). Mouse monoclonal antibodies against HRV VP2 were obtained from QED Bioscience (San Diego, CA, USA).

Song J., Chi M., Luo X., Song Q., Xia D., Shi B, & Han J. (2019). Non-Structural Protein 2B of Human Rhinovirus 16 Activates Both PERK and ATF6 Rather Than IRE1 to Trigger ER Stress. Viruses, 11(2), 133.

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Western Blot Analysis of ER Stress Markers

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EDL muscle samples from F2 female rats were processed as described previously [17] (link). An equal amount of protein (15 μg) was separated by SDS-PAGE using Criterion XT Precast gels (Bio-Rad; Hercules, CA) and transferred to polyvinylidene difluoride membrane (PVDF) (Immobilon-P, Millipore; Billerica, MA). Protein content was determined using the following antibodies: ATF6α-p50 (#sc-22799) and CHOP (#sc-7351) from Santa Cruz Biotechnology (California, USA), GRP78 (#ab21685), GRP94 (#ab90458), p-IRE1α (#ab48187) and XBP1 (#ab37152) from Abcam (Cambridge, UK) and p-EIF2A (#9721) and p-JNK (#9251) from Cell Signaling Technology (Massachusetts, USA). Antibody dilutions were performed according to manufacturer's instructions. Ponceau S staining of the PVDF membranes was used for normalization.

Alm P.S., de Castro Barbosa T., Barrès R., Krook A, & Zierath J.R. (2017). Grandpaternal-induced transgenerational dietary reprogramming of the unfolded protein response in skeletal muscle. Molecular Metabolism, 6(7), 621-630.

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Western Blot Analysis of Membrane Proteins

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Cells were lysed with Cell Lysis Buffer (Cell Signaling Technology) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). For Western blot to confirm expression of membrane-localized CD4 fusion protein in Fig. 5B, cells were lysed in RIPA buffer made using recipe found here: https://www.abcam.com/protocols/general-western-blot-protocol#Solutions%20and%20reagents. Lysates were mixed with NuPAGE LDS Sample Buffer (Life Technologies) and NuPAGE Sample Reducing Agent (Life Technologies) and were run on Bolt 4–12% Bis-Tris Plus gels (Invitrogen). Gels were transferred with iBlot2 PVDF Mini Stacks (Invitrogen) and iBlot2 (Invitrogen). Blots were probed with antibodies for mCherry (GTX128508, GeneTex), GFP (sc-99G, Santa Cruz Biotechnology), Tid1 (EPR12414, abcam or RS-11, Santa Cruz Biotechnology), p53 (DO-1, Santa Cruz Biotechnology), CHOP (9C8, Novus Biologicals), ATF6-N (70B1413.1, Novus Biologicals), p EIF2a (9721, Cell Signaling Technology), EIF2a (9722, Cell Signaling Technology), Bax (6A7, Santa Cruz Biotechnology), MDM2 (sc-965, Santa Cruz Biotechnology), Cyclophillin D (E11AE12BD4, abcam), Hsp90 (C45G5, Cell Signaling Technology), Cox IV (3E11, Cell Signaling Technology), and GAPDH (D4C6R, Cell Signaling Technology). Blots were imaged using Bio-Rad ChemiDoc MP Imaging System or Azure Biosystems Sapphire Biomolecular Imager.

Preston A.J., Rogers A., Sharp M., Mitchell G., Toruno C., Barney B.B., Donovan L.N., Bly J., Kennington R., Payne E., Iovino A., Furukawa G., Robinson R., Shamloo B., Buccilli M., Anders R., Eckstein S., Fedak E.A., Wright T., Maley C.C., Kiso W.K., Schmitt D., Malkin D., Schiffman J.D, & Abegglen L.M. (2023). Elephant TP53-RETROGENE 9 induces transcription-independent apoptosis at the mitochondria. Cell Death Discovery, 9, 66.

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Western Blot Analysis of Cellular Markers

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Western blotting was conducted as previously described 34 (link). The following primary antibodies were used in this study: GRP78 (# 3177; Cell Signaling Technology), GRP94 (#3196-1; Epitomics), pERK1/2 (#sc-377400, Santa Cruz Biotechnology), p65 (#sc-8008; Santa Cruz Biotechnology), CHOP(#2895; Cell Signaling Technology), cleaved PARP (#sc-56196, Santa Cruz Biotechnology), NLRP3 (#sc-134306; Santa Cruz Biotechnology), HXK2 (#sc-374091, Santa Cruz Biotechnology), IL-1β (#sc-12742; Santa Cruz Biotechnology), IL-18 (#sc133127; Santa Cruz Biotechnology), p-eIF2a (#33981; Cell Signaling Technology), Casp1 (#YH050707C; Epitonics), ZDHHC1 (#AP10315a; Abgent), E-cadherin (#sc8426; Santa Cruz Biotechnology), Occludin (#TA306787; Origene), N-cadherin (#610920; BD transduction laboratories), Vimentin (#5741s; Cell Signaling Technology), G6PD (#12263s; Cell Signaling Technology), Casp3 (#9664; Cell Signaling Technology), Casp7 (#8438; Cell Signaling Technology), GLUT1(#sc-377228; Santa Cruz Biotechnology), beta-actin (#sc8432; Santa Cruz Biotechnology), GAPDH (#sc-47727; Santa Cruz Biotechnology).

Le X., Mu J., Peng W., Tang J., Xiang Q., Tian S., Feng Y., He S., Qiu Z., Ren G., Huang A., Lin Y., Tao Q, & Xiang T. (2020). DNA methylation downregulated ZDHHC1 suppresses tumor growth by altering cellular metabolism and inducing oxidative/ER stress-mediated apoptosis and pyroptosis. Theranostics, 10(21), 9495-9511.

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Characterizing Apoptotic Signaling in MIN6 Cells

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MIN6 cells were lysed in cell lysis buffer containing 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 10 mm dithiothreitol, 1 mm Na₃VO₄, and complete protease inhibitor mixture. Equal amounts of protein were resolved by 10% or 4–12% NuPAGE (Invitrogen) gels, transferred onto PVDF membrane (Millipore), and blots were probed with antibodies against IER3IP1 (sc-84849; Santa Cruz), Puma (7467; Cell Signaling Technologies), Bim (202000; Calbiochem), cleaved caspase-3 (9661; Cell Signaling Technologies), p-FoxO1/Fox3a (9461; Cell signaling), FoxO1 (2880; Cell signaling), Fox3a (2497; Cell signaling), β-actin (A-2066; Sigma), Bax (6A7) (2281-MC-100; Trevigen), Bax (N20) (sc-493, Santa Cruz), Bak (upstate, 06536), p-IRE1α (NB100-2323; Novus), IRE1α (3294; Cell signaling), p-PERK (3179, Cell signaling), PERK (sc-9477; Santa Cruz), ATF4 (ARP37017_P050, Aviva systems biology); p-eIF2a (9721, Cell signaling), eIF2a (9722; Cell signaling), and Chop (2895; Cell signaling). To detect BAX activation, immunoprecipitation (IP) was performed using 1% CHAPS buffer (1% CHAPS, 142.5 mM KCl, 2 mM CaCl₂, 20 mM Tris-Cl, pH 7.4). Anti-6A7 IP was performed using 1% CHAPS buffer. Antibody detection was accomplished using enhanced chemiluminescence (PERKinElmer) and LAS-3000 Imaging system (FUJIFILM).

Sun J, & Ren D. (2017). IER3IP1 deficiency leads to increased β-cell death and decreased β-cell proliferation. Oncotarget, 8(34), 56768-56779.

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Western Blot Analysis of ER Stress Markers in Ts65Dn Mouse Model

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For Western blot, 30 µg of proteins (3, 9, and 18mo Ts65Dn and Euploid) were separated by 7.5% and 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), using Criterion TGX (Tris-Glycine extended) Stain-Free precast gels. Blots were normalized on gel total protein load acquired taking advantage of the Stain free technology on ChemiDoc XP densitometer (Bio-Rad). After image acquisition gels were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 7% nonfat dry milk (Lab Scientific) with 0.02% sodium azide and incubated over night at 4 °C with following primary antibodies: p PERK (1:250) and PERK(1:250), CHOP(1:500), GRP78 (1:500), ATF6 (1:500), pIRE1 (1:500), IRE1 (1:500) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), p eIF2a (1:500), and eIF2a (1:1000) from Cell Signaling). The membranes were incubated for 1 h at room temperature with secondary antibody horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG (1:5000); Sigma-Aldrich, St. Louis, MO, USA). Membranes were developed with the Clarity Western ECL Substrate (Bio-Rad), visualized using ChemiDoc XP (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).

Lanzillotta C., Tramutola A., Meier S., Schmitt F., Barone E., Perluigi M., Di Domenico F, & Abisambra J.F. (2018). Early and Selective Activation and Subsequent Alterations to the Unfolded Protein Response in Down Syndrome Mouse Models. Journal of Alzheimer's disease : JAD, 62(1), 347-359.

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Immunohistochemical Analysis of OS Proteins

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For immunohistochemical assessments, 4-μm-thick paraffin-embedded OS sections were mounted onto coated glass slides to detect the proteins under investigation. Following antigen retrieval and blocking of endogenous peroxidases and nonspecific protein binding, the slide sections were incubated first with primary antibodies against p-eIF2a, P-JNK, LC3 (diluted 1:200) purchased from Cell Signaling Technology, followed by appropriate horseradish peroxidase-conjugated secondary antibodies.

Oh J.Y., Lee Y.J., Sai S., Ohno T., Kong C.B., Lim S.H, & Kim E.H. (2020). The Unfolded Protein Response: Neutron-Induced Therapy Autophagy as a Promising Treatment Option for Osteosarcoma. International Journal of Molecular Sciences, 21(11), 3766.

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Western Blotting Analysis of Signaling Pathways

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Total cell lysates were prepared and analyzed by western blotting as described previously [32 (link)]. The proteins were recovered in sodium dodecyl sulfate (SDS) buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. The membrane was blotted with appropriate primary and secondary antibodies. Rabbit polyclonal antibodies against ULK1, p-ULK1^S757, FLT3, p-FLT3, STAT5, p-STAT5, p-MEK, p-ERK, caspase-9, caspase-3, PARP, AMPKα, p-AMPKα^T172, mTOR, p-mTOR^S2448, p70S6K, p-p70S6K^T389, PERK, and p-eIF2a were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies against LC3 and p-ATG13^S318 were obtained from Novus Biologicals (Littleton, CO, USA). Mouse anti-p62/SQSTM antibodies were procured from Abnova (Taipei, Taiwan). Anti-p-PERK^T982 antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-p-ULK1^S555 and mouse anti-α-tubulin monoclonal antibodies were obtained from Merck Millipore (Billerica, MA, USA). The secondary antibodies were coupled to horseradish peroxidase. The blots were visualized using an enhanced chemiluminescence (GE Healthcare Bio-Sciences; RPN2232).

Hwang D.Y., Eom J.I., Jang J.E., Jeung H.K., Chung H., Kim J.S., Cheong J.W, & Min Y.H. (2020). ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia. Journal of Experimental & Clinical Cancer Research : CR, 39, 85.

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