Atf4 sc 200

Manufactured by Santa Cruz Biotechnology

Sourced in United States

ATF4 (sc-200) is an antibody product offered by Santa Cruz Biotechnology. It is a research-use antibody that recognizes the ATF4 protein. ATF4 is a transcription factor involved in the cellular stress response. The antibody can be used to detect and study the ATF4 protein in various experimental applications.

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10 protocols using atf4 sc 200

Immunoblotting Quantification of Cellular Signaling

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Protein lysates were separated on polyacrylamide gels followed by transfer onto nitrocellulose membranes. Membranes were incubated with antibodies against phospho-ACC (Ser79) (#3661), phospho-AMPK (Thr172) (#2531), phospho-eIF2α (Ser51) (#3398), phospho-cJun (Ser73) (#3270), phospho-S6 ribosomal protein (Ser240/244) (#2215), ACC (#3662), AMPK (#2532) (Cell Signalling Technology), sXBP1 (6196, BioLegend), phospho-IRE1 (Ser724) (ab124945, Abcam), ATF6 (NBP1-75478, Novus Biologicals), ATF4 (sc-200), or nucleolin (sc-13057) (Santa Cruz Biotechnology) followed by IRDye 800-coupled secondary antibodies (LICOR Biosciences). Blots were visualized and quantified using the Odyssey imaging system (LICOR Biosciences).

Boß M., Newbatt Y., Gupta S., Collins I., Brüne B, & Namgaladze D. (2016). AMPK-independent inhibition of human macrophage ER stress response by AICAR. Scientific Reports, 6, 32111.

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Antibody Sourcing for Western Blot

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The powder of BIX-01294 (B9311) trihydrochloride and EHMT2 (G6919) antibody were purchased from Sigma-Aldrich (City of Saint Louis, Missouri, US). CASP3 (IMG-145) antibody was purchased from Imgenex. USP9X (5751), DDIT3 (2895), CASP8 (9746), CASP9 (9502), PARP1 (9542), HSPA5 (3183) and ERN1 (3294) antibodies were purchased from Cell Signaling Technology (Boston, Massachusetts, US). ATF4 (SC-200) and MCL1 (SC-12756) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, US). PMAIP1 (OP180) antibody was purchased from Calbiochem (San Diego, California, US).

Cui J., Sun W., Hao X., Wei M., Su X., Zhang Y., Su L, & Liu X. (2015). EHMT2 inhibitor BIX-01294 induces apoptosis through PMAIP1-USP9X-MCL1 axis in human bladder cancer cells. Cancer Cell International, 15, 4.

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Western Blot Analysis of Cellular Stress Markers

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Cells were lysed in lysis buffer consisting of 50 mM HEPES, 40 mM NaCl, 2 nM EDTA, 0.5% Triton X-100, 1.5 mM Na₃VO₄, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium beta-glycerophosphate and 1 tablet of protease inhibitor. Cell lysates were separated on a 4–20% Tris-gel, transferred to nitrocellulose membrane and blocked in PBST +5% milk for an hour. Membranes were incubated with primary antibody at the following concentrations: ATF4 (sc-200; Santa Cruz, Santa Cruz, CA) 1:100, phosphor-eIF2α (5324; Cell Signaling, Danvers, MA) 1:200, eIF2α (3597; Cell Signaling) 1:1000, p-PERK 1:200 (sc-32577; Santa Cruz), PERK 1:200 (sc-13073; Santa Cruz), BiP (3177; Cell Signaling) 1:1000, eIF4E (610270; BD Biosciences, San Jose, CA) 1:1000 in PBS + 5% BSA overnight at 4°C. Next day washed and incubated with 1:2000 HRP-conjugated secondary before development with SuperSignal West Pico Substrate (#34080; Pierce, Rockford, IL).

Dixon S.J., Patel D.N., Welsch M., Skouta R., Lee E.D., Hayano M., Thomas A.G., Gleason C.E., Tatonetti N.P., Slusher B.S, & Stockwell B.R. (2014). Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosis. eLife, 3, e02523.

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Antibody Panel for Cell Signaling

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eIF2α, P-eIF2α (Ser51), Total S6K1, P-S6K1 (T389), Cleaved PARP, total and cleaved caspase-3 antibodies were purchased from Cell Signaling Technologies, ATF4 (SC-200) from Santa Cruz and MAP1LC3B antibody from Novus. Antibody Puromycin (clone 12D10) was kindly given by Phillipe Pierre (Centre d'Immunologie de Marseille-Luminy, France).

Mesclon F., Lambert-Langlais S., Carraro V., Parry L., Hainault I., Jousse C., Maurin A.C., Bruhat A., Fafournoux P, & Averous J. (2017). Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells. Oncotarget, 8(16), 27440-27453.

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Western Blot of Apoptosis and UPR Markers

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Samples were separated on 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA). For cytoplasmic fractions or whole cell lysates, 100 μg of total protein was loaded. For nuclear fractions, 10 μg of total protein was loaded. The following antibodies and conditions were used: cleaved PARP (9544; 1:500; Cell Signaling, Danvers, MA), caspase-6 (9762; 1:500; Cell Signaling), cleaved caspase-3 (9664; 1:500; Cell Signaling), XBP-1 (sc-7160; 1:200; Santa Cruz, Dallas, TX), ATF4 (sc-200; 1:200; Santa Cruz), ATF6 (IMG-273; 1:200; Imgenex, San Diego, CA); LRH-1 (PP-H2325-10; 1:200; Perseus Proteomics, Tokyo, Japan), pATF2 (9225S; 1:500; Cell Signaling [note: we have experienced difficulties with recent batches]), ATF2 (ab47476; 1:1000; Abcam, Cambridge, England); and PLK3 (4896S; 1:400; Cell Signaling). Immobilon Western substrate (Millipore, Billerica, MA) was used for detection.

Mamrosh J.L., Lee J.M., Wagner M., Stambrook P.J., Whitby R.J., Sifers R.N., Wu S.P., Tsai M.J., DeMayo F.J, & Moore D.D. (2014). Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution. eLife, 3, e01694.

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Antibodies for Endoplasmic Reticulum Stress

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Antibodies for C/EBP homologous protein (CHOP) (#2895), cleaved caspase-3 (#9661), cyclin-D1 (#2926), EGFR (#2232), eIF2α (#9722), and phospho-eIF2α (Ser51) (#9721), protein kinase R-like ER kinase (PERK) (#3192) were purchased from Cell Signaling Technology (Danvers, MA, USA), and that for KDEL sequence (ADI-SPA-827-F) was from Enzo Life Sciences (Loerrach, Germany). Anti-activating transcription factor 4 (ATF4) (sc-200) and β-actin (AC-15) antibodies were products of Santa Cruz Biotechnology (Dallas, TX, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

Koyama S., Omura T., Yonezawa A., Imai S., Nakagawa S., Nakagawa T., Yano I, & Matsubara K. (2015). Gefitinib and Erlotinib Lead to Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Independent of Epidermal Growth Factor Receptor in A549 Cells. PLoS ONE, 10(8), e0136176.

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Western Blot Analysis of Cellular Protein Extracts

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Isolation of total and nuclear extracts was performed as described elsewhere [28] (link). Proteins (30 μg) were separated by SDS-PAGE on 10% acrylamide separation gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore). Western blot analysis was performed using antibodies against VLDLR (sc-18824), Nrf2 (sc-722), Nqo1 (sc-393736), ATF4 (sc-200) (Santa Cruz), VLDLR (AF2258) (R&D system), eIF2α (9722), phospho-eIF2α (Ser51) (9721), IgG control (2729S) (Cell Signaling Technology Inc., Danvers, MA), actin (A5441) (Sigma–Aldrich, Madrid, Spain). Detection was achieved using the Western Lightning® Plus-ECL chemiluminescence kit (PerkinElmer, Waltham, MA, USA). The equal loading of proteins was assessed by Ponceau S staining. The size of detected proteins was estimated using protein molecular-mass standards (Bio-Rad).

Zarei M., Barroso E., Palomer X., Dai J., Rada P., Quesada-López T., Escolà-Gil J.C., Cedó L., Zali M.R., Molaei M., Dabiri R., Vázquez S., Pujol E., Valverde Á.M., Villarroya F., Liu Y., Wahli W, & Vázquez-Carrera M. (2017). Hepatic regulation of VLDL receptor by PPARβ/δ and FGF21 modulates non-alcoholic fatty liver disease. Molecular Metabolism, 8, 117-131.

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Western Blot Protein Analysis Protocol

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Protein extracts were prepared by using 1 × RIPA buffer (20–188, Millipore) supplemented with protease (1860932, ThermoFisher) and phosphatase inhibitors (78428, ThermoFisher). For analysis of transcriptional regulators, lysis buffer was supplemented with 1% SDS and benzonase (70746–4, EMD Millipore). Equal amounts of total protein were separated on NuPAGE Bis-Tris or Tris-Acetate (for large proteins) gels (Life Technologies), transferred to nitrocellulose membranes and subjected to Western blotting with indicated primary antibodies. The following primary antibodies were used: phospho-Thr899 GCN2 (ab75836, Abcam), total GCN2 (3302, Cell Signaling), phospho-Thr-389-S6K1 (9234, Cell Signaling), S6K1 (2708, Cell Signaling), vinculin (V9131, Sigma-Aldrich), ATF4 (sc-200, Santa Cruz), MED12 (A300-774A, Bethyl), TBP (A301-229A, Bethyl), CBP (A300-362A, Bethyl), CBFα1 (12556, Cell Signaling), BRD4 (ab128874, Abcam), CTCF (A700-041-T, Bethyl), RNA pol II (39497, Active Motif), Histone H3 (3638, Cell Signaling), α-Tubulin (T9026, Sigma-Aldrich), β-Actin (A5441, Sigma-Aldrich), HA tag (2367, Cell Signaling), puromycin (MABE343, EMD Millipore), and GLS (ab156876, Abcam).

Pavlova N.N., King B., Josselsohn R.H., Violante S., Macera V.L., Vardhana S.A., Cross J.R, & Thompson C.B. (2020). Translation in amino-acid-poor environments is limited by tRNAGln charging. eLife, 9, e62307.

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Pemetrexed-Induced Apoptosis Mechanism

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Pemetrexed was purchased from Toronto Research Chemicals, Inc. (Toronto, ON, Canada). Caspase-3 antibody (clone: 31A1067) was obtained from Imgenex (San Diego, CA, USA). Caspase-9 (cat. no. 9502s), PARP (cat. no. 9542L) and Usp9x (cat. no. 5751s) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mcl-1 (cat. no. sc-12756), ATF4 (sc-200) and ATF3 (sc-81189) antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA). Puma (clone no.114C307) and β-actin (clone no. AC-15) antibodies were purchased from Sigma (St. Louis, MO, USA). Noxa (clone no.114c307) antibody was purchased from EMD Millipore (Billerica, MA, USA).

Yan J., Zhong N., Liu G., Chen K., Liu X., Su L, & Singhal S. (2014). Usp9x- and Noxa-mediated Mcl-1 downregulation contributes to pemetrexed-induced apoptosis in human non-small-cell lung cancer cells. Cell Death & Disease, 5(7), e1316-.

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Western Blot Analysis of Liver Proteins

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Liver tissue was homogenized in Tissue Protein Extraction Reagent (T-PER®, Waltham, Massachusetts, USA). Equal amounts of protein were loaded onto Criterion Tris-HCl gels (Bio-Rad Lab, Hercules, CA) and electro-transferred to Immobilion® - FL PVDF membrane (EMD Millipore). The membranes were blocked in either 5% Non-Fat Dry Milk or 5% bovine serum albumin in TBST depending on the primary antibody manufacturer’s recommendation for 1 hour at room temperature. Membranes were then incubated with the primary antibody overnight followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour. Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, Massachusetts, USA) was used to detect the HRP activity of the secondary antibodies and expose to films. Primary antibodies used were: phopsho-PERK (Product # 3179), total PERK (Product # 3192), phospho-eIF2α (9721), and total eIF2α (9722) from Cell Signaling Technology, ATF4 (sc-200) and CHOP (sc-7351) from Santa Cruz Biotechnology, GAPDH (Millipore, Mab374) and alpha-tubulin (Sigma, T9026).

Bandla H., Dasgupta D., Mauer A.S., Nozickova B., Kumar S., Hirsova P., Graham R.P, & Malhi H. (2018). Deletion of endoplasmic reticulum stress-responsive co-chaperone p58IPK protects mice from diet-induced steatohepatitis. Hepatology research : the official journal of the Japan Society of Hepatology, 48(6), 479-494.

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