Anti aif

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-AIF is a laboratory reagent that detects the Apoptosis-Inducing Factor (AIF) protein. AIF is a mitochondrial protein that plays a role in programmed cell death. This antibody can be used to identify and study the AIF protein in various biological samples.

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16 protocols using anti aif

Apoptosis Induction and Mitochondrial Dysfunction

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The following reagents and antibodies were used: Bongkrekic acid (Santa Cruz Biotechnology, La Jolla, CA), JC-1 (eBioscience, San Diego, CA), Cyclosporine A, 2′,7′-dichlorofluorescein acetate (DCFH-DA), N-acetyl cysteine (Sigma, St.Louis, MO), Dioleylphosphatidylserine (Avanti Lipids, Alabaster, AL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) ((Roche Diagnostics, Indianapolis, IN), and disuccinyl suberate (Thermo Scientific Fischer, Rockford, IL). Anti-Bcl-2, anti-β-Actin (Abcam, Cambridge, MA), anti-Cyto c (eBioscience, San Diego, CA), anti-AIF, anti-caspase-3, anti-cleaved caspase-3 (Cell Signaling Technology, Boston, MA, anti-Survivin, Smac/Diablo, α-Tubulin (Novus biological, Littleton, CO), anti-COX-4, anti-Bax (N-20; Santa Cruz Biotechnology, La Jolla, CA), anti-Bax (polymer-recognizing A67 clone; Sigma, St.Louis, MO) and anti- cleaved PARP (Millipore, Bedford, MA).
Animal maintenance and experimental procedures were carried out in accordance with the US National Institute of health Guidelines for Use of Experimental Animals and approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and Cincinnati Children's Hospital Medical Center.

Sulaiman M.K., Chu Z., Blanco V.M., Vallabhapurapu S.D., Franco R.S, & Qi X. (2015). SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas. Molecular Cancer, 14, 78.

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Investigating b-AP15-Induced Cell Death

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b-AP15 was obtained from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO at a stock concentration of 10 mM, aliquoted and stored at −80°C. Other agents are N-acetyl-L-cysteine (NAC, Sigma-AldrichInc., St. Louis, MO); pan caspase Inhibitor Z-VAD-FMK (EnzoLife Sciences International, Inc, Plymouth Meeting, PA). Antibodies (Abs) used in this study were purchased from following sources: anti-CDK2, CDK4, CDK6, anti-cyclinD1, anti-p27, anti-PARP, anti-Bcl-2 (50E3), anti-Bim (Y36), anti-Noxa (D8L7U), anti-BIP (C50B12), anti-CHOP (L63F7), eIF2α, phospho-eIF2α (Ser51), anti-HSP70, anti-HSP90, anti-AIF, cytochrome C and anti-Phospho-MDM2 (Cell Signaling Technology, Beverly, MA, USA); anti-Rb, phospho-Rb (S780), anti-cleaved caspase-8 (Cleaved Asp384) (Assay biotechnology Company, Inc); anti-cleaved caspase-9 p35 (D315), anti-cleaved caspase-3 (p17), anti-AR, anti-MDM2 and anti-GAPDH (Bioworld Technology, Inc). anti-p53(Abcam), anti-ubiquitin (P4D1), anti-K48-linked tetra-ubiquitin, Bax (B-9) (Santa Cruz Biotechnology, Santa Cruz, CA); MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). CCK-8 assay kit, PI and Annexin V-FITC apoptosis Detection Kit, DCFH-DA and cell apoptosis Rhodamine 123 Detection Kit were purchased from Keygen Company (Nanjing, China). Dynabeads antibody coupling kit was from Life technologies.

Cai J., Xia X., Liao Y., Liu N., Guo Z., Chen J., Yang L., Long H., Yang Q., Zhang X., Xiao L., Wang X., Huang H, & Liu J. (2017). A novel deubiquitinase inhibitor b-AP15 triggers apoptosis in both androgen receptor-dependent and -independent prostate cancers. Oncotarget, 8(38), 63232-63246.

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Protein Lysates Western Blot Analysis

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Protein lysates were generated, and western [28 (link)] blots performed the following standard procedures [18 (link)]. All primary antibodies used in western blot analyses [anti-γH2AX, anti-phospho-CHK1 (Ser345), anti-phospho-CHK2 (Thr68), anti-phospho-BRCA1 (1524), anti-phospho-ATM (Ser 1981), anti-phospho-ATR (Ser 428), anti-p53, anti-acetyl-histone 3, anti-acetyl-histone 4, anti-Bcl-XL, anti-Mcl1, anti-Bcl2 and anti-AIF] were obtained from Cell Signaling, Boston, MA, USA. Horseradish peroxidase linked-donkey (anti-rabbit), sheep (anti-mouse), or mouse (anti-goat) immunoglobulins were used as secondary antibodies at a 1:5000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

López-Iglesias A.A., Herrero A.B., Chesi M., San-Segundo L., González-Méndez L., Hernández-García S., Misiewicz-Krzeminska I., Quwaider D., Martín-Sánchez M., Primo D., Paíno T., Bergsagel P.L., Mehrling T., González-Díaz M., San-Miguel J.F., Mateos M.V., Gutiérrez N.C., Garayoa M, & Ocio E.M. (2017). Preclinical anti-myeloma activity of EDO-S101, a new bendamustine-derived molecule with added HDACi activity, through potent DNA damage induction and impairment of DNA repair. Journal of Hematology & Oncology, 10, 127.

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Immunohistochemical Analysis of PHB Proteins

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IHC stainings were performed as previously described [32 (link)] using the Envision Kit (Dako Denmark A/S). Rabbit anti-PHB1, anti-PHB2, anti-AIF antibodies were purchased from Cell Signaling Technology and mouse anti-Ki67 was obtained from Dakko. Mouse and rabbit isotypic control antibodies were purchased from Sigma. For PHB expression in biopsy samples, the staining intensity was scored from 0 to 4 as follows: 0, negative; 1, weak; 2, moderate; 3, strong; 4, very strong. All tumor samples were PHB1 positive. For statistical analysis, a staining index score ≥ 3 indicated tumors with high PHB1 or PHB2 expression, and < 3 low PHB1 or PHB2 expression.

Bentayeb H., Aitamer M., Petit B., Dubanet L., Elderwish S., Désaubry L., de Gramont A., Raymond E., Olivrie A., Abraham J., Jauberteau M.O, & Troutaud D. (2019). Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects. Journal of Experimental & Clinical Cancer Research : CR, 38, 450.

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Immunofluorescence Staining of Cell Proteins

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Cells were incubated in primary antibody (anti-NPL4, 1:1 000; Novus Bio; anti-AIF, 1:1 000; Cell Signaling; anti-phospho-Histone H2A.X (Ser139) 1:500, Merk-Millipore) after treatment and fixation, and stained with the secondary antibody (Jackson Laboratory, Bar Harbor, ME, USA), and DAPI (Invitrogen, Carlsbad, CA, USA). Slides were prepared with mounting medium (Dako, Santa Clara, CA, USA) and imaged with a Zeiss microscope. Signal intensity was quantified with ImageJ (National Institute of Health).

Serra R., Zhao T., Huq S., Gorelick N.L., Casaos J., Cecia A., Mangraviti A., Eberhart C., Bai R., Olivi A., Brem H., Jackson E.M, & Tyler B. (2021). Disulfiram and copper combination therapy targets NPL4, cancer stem cells and extends survival in a medulloblastoma model. PLoS ONE, 16(11), e0251957.

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Polyphenol-Mediated Signaling Pathway Analysis

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At various times (24, 48 and 72 h) after 5 µg/ml of peel polyphenols treatment, the LS174 cells were collected and lysed at room temperature with 100 µl of Laemmli buffer (1×). Protein content of the cell lysates was quantified using the BCA method (Bicinchoninic Acid Protein Assay kit, Sigma). Equal amounts of protein (30 µg/sample) were separated electrophoretically by 10 % SDS-PAGE and blotted onto PVDF membranes (Immobilon-Millipore). The blots were probed with primary antibodies and incubated with a horseradish peroxidase-conjugated anti-IgG in a blocking buffer for 1 h. Primary antibodies anti-phospho-AKT, anti-AKT, anti-ERK₂, anti-phospho-JNK/SAPK, anti-JNK/SAPK, anti-phospho-p38, anti-phospho-IKKα/β, anti-IKKα, ECLhCZIBPBHhf-iFeN8r/" target="_blank">anti-PARP, anti-caspase 9, anti-caspase 3, anti-AIF, anti-Cyclin D1 and loading control anti-actin were from cell Signaling Technology (Danvers, MA, USA). Anti-phospho ERKs were from Sigma-Aldrich (L’Isle d’Abeau, Chesnes, France), and anti-horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were from Promega (Madison, WI). After washing, the blots were developed with enhanced chemiluminescence (ECL) (Millipore) and exposed to X-ray film.

Riahi-Chebbi I., Haoues M., Essafi M., Zakraoui O., Fattouch S., Karoui H, & Essafi-Benkhadir K. (2016). Quince peel polyphenolic extract blocks human colon adenocarcinoma LS174 cell growth and potentiates 5-fluorouracil efficacy. Cancer Cell International, 16, 1.

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Immunofluorescence Assay for EGFR, AIF, and Phosphatidylserine

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3x10⁴ cells were cultured on coverslips, treated with HT (10-100 μM), cetuximab (1-10 μg/ml) with/without EGF (5 ng/ml) for 8-24 or 48 h (with treatment of HT every 24 h) and then fixed in paraformaldehyde (4%, 10 min), washed in PBS and permeabilized, if necessary, with Triton X-100 (0.5% in PBS containing 0.5% BSA) and then incubated with BSA (45 min). Cells were then incubated for 16 h with anti-EGFR (1:40), anti-AIF (1:50, Cell Signaling), anti-phosphatedylserine (1:50, 05-719 Merk Millipore), anti-occludin (1:80, 71-1500 ZYMED) diluted in PBS containing 0.5% BSA. To investigate phosphatedylserine externalization on cell membranes, cells were fixed in paraformaldehyde (4%, 10 min), washed in PBS, incubated with BSA (45 min) without permeabilization, and then incubate for 16 h with anti-phosphatedylserine (1:50, 05-719 Merk Millipore) diluted in PBS containing 0.5% BSA. After incubation with the secondary antibody ALEXAFLUOR 488 anti-mouse or anti-rabbit (A11001, A11011 respectively; 1:200 1 h, Thermo Fisher), cells were washed and incubated with DAPI (1 μg/mL, 20 min), washed and the coverslips were mounted in Fluoromount Aqueous Mounting Medium.

Terzuoli E., Nannelli G., Frosini M., Giachetti A., Ziche M, & Donnini S. (2017). Inhibition of cell cycle progression by the hydroxytyrosol–cetuximab combination yields enhanced chemotherapeutic efficacy in colon cancer cells. Oncotarget, 8(47), 83207-83224.

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Western Blot Protein Expression Analysis

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The antibodies (anti-HSPA8, anti-AIF, anti-14.3.3 ζ/δ, anti-Calreticulin, anti-HMGB1, anti-HPRT1 and Anti-PP2A A subunit) used in this study were from Cell Signaling Technology and were used as recommended by the supplier. Cell lysates were prepared from all available cell lines. After PBS washing, the pellets were re-suspended in Protein Extraction Reagent Type 4, and following sonication in ice, the lysates were clarified by centrifugation. The protein contents were measured using the Bradford assay and 20 μg of each sample was used for SDS PAGE separation and subsequent transferred on Hybond-C membranes (GE Healthcare Life Sciences). Primary antibodies were detected by secondary HRP-conjugated antibodies using the ECL detection system (Advansta). Whole lane normalization was used for quantitative investigation, as previously described by Colella et al. [23 (link)] and Gürtler et al. [24 (link)]. Experiments were performed in quintuplicates and the statistical analysis was performed using the paired t-test.

Menotta M., Orazi S., Gioacchini A.M., Spapperi C., Ricci A., Chessa L, & Magnani M. (2018). Proteomics and transcriptomics analyses of ataxia telangiectasia cells treated with Dexamethasone. PLoS ONE, 13(4), e0195388.

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Western Blot Analysis of Protein Fractions

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After drug treatment, cytosolic and nuclear lysates were extracted with the NE-PER Nuclear Protein Extraction Kit (Millipore, Bedford, MD, USA) according to the manufacturer’s protocol. The cytosolic fraction was extracted with cytoplasmic lysis buffer (1× cytoplasmic lysis buffer, 0.5 mM DTT, 1:1000 dilution of inhibitor cocktail) while the nuclear fraction was extracted with nuclear extraction buffer (1× nuclear extraction buffer, 1:1000 dilution of inhibitor cocktail). Protein concentrations were determined with the Bio-Rad Dc protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then probed with primary antibodies (anti-AIF) (Cell Signaling, Beverly, MA, USA) and subsequently incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG) (Invitrogen). After washing with 0.1% TBS-T (USB), the membrane was visualized by an enhanced chemiluminescence detection system (Bio-Rad). For the cytosolic fraction, protein expression was compared with β-actin (Sigma-Aldrich). For the nuclear fraction, lamin A/C (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for normalization.

Law C.K., Kwok H.H., Poon P.Y., Lau C.C., Jiang Z.H., Tai W.C., Hsiao W.W., Mak N.K., Yue P.Y, & Wong R.N. (2014). Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor. Chinese Medicine, 9, 11.

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Western Blot Antibody Optimization Protocol

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Monoclonal anti-Caspase-12 antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-cleaved caspase-3, anti-PUMA, anti-HistonH2A, anti-AIF, anti-RIP1, anti-RIP3, anti-MLKL, anti-p53, anti-COX IV and anti-Lamin B were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-RPE65 (specific for RPE cells) was from Abcam (Cambridge, MA, USA). The preparation and use of FITC-labeled anti-MCMV EA and biotin-anti-EA has been described previously [24 (link)]. Anti-β-actin was from Sigma-Aldrich Corp. (St. Louis, MO, USA). Texas red avidin D, DyLight 594 goat anti-rabbit IgG antibody and DyLight 488 horse anti-mouse IgG antibody were from Vector Laboratories, Inc. (Burlingame, CA, USA). Goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). BCA assay kit, NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents (78833) and Mitochondria Isolation Kit for Tissue (89801) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). TUNEL assay kit was from Roche (Indianapolis, IN, USA).

Zhang X., Xu J., Marshall B., Dong Z., Smith S.B, & Zhang M. (2021). The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis. International Journal of Molecular Sciences, 22(15), 8135.

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