Anti bak

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-Bak is a primary antibody that targets the Bak protein. Bak is a pro-apoptotic member of the Bcl-2 protein family and plays a key role in the intrinsic apoptosis pathway. Anti-Bak can be used to detect and quantify Bak expression in various cell and tissue samples.

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14 protocols using anti bak

Evaluating Mcl-1 Interactions with Bim and Bak

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Interactions between Mcl-1 and Bim or Bak were evaluated by co-IP analysis. CHAPS buffer (150 mmol/L NaCl, 10 mmol/L HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid] pH 7.4, protease inhibitors, and 1% CHAPS) was employed to avoid artifactual associations [43] (link). Cells were lysed in CHAPS buffer and 200 µg of protein per condition were immunoprecipitated with 1 µg anti-Mcl-1 (Santa Cruz Biotechnology or BD Biosciences), anti-Bak, or anti-Bim (Santa Cruz Biotechnology), followed by Dynabeads (Dynal, Oslo, Norway). IP samples were then subjected to Western blot analysis using anti-Bim (Millipore), anti-Mcl-1, or anti-Bak (Santa Cruz Biotechnology) as primary antibodies, respectively.
To monitor Bak and Bax conformational change, anti-Bax (6A7, Sigma) or anti-Bak (Ab-1, Millipore) antibodies, which only recognize Bax or Bak that have undergone conformational change, were used for IP, followed by Western blot analysis using anti-Bax and anti-Bak as primary antibodies.

Pei X.Y., Dai Y., Felthousen J., Chen S., Takabatake Y., Zhou L., Youssefian L.E., Sanderson M.W., Bodie W.W., Kramer L.B., Orlowski R.Z, & Grant S. (2014). Circumvention of Mcl-1-Dependent Drug Resistance by Simultaneous Chk1 and MEK1/2 Inhibition in Human Multiple Myeloma Cells. PLoS ONE, 9(3), e89064.

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TRAIL-induced Apoptosis Regulation Mechanisms

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Cannabidiol and VAS2870 were purchased from Sigma (St. Louis, MO, USA). TRAIL and anti-DR5 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-Bak, anti-Bcl-2, anti-Mcl-1, anti-Bcl-xL, and anti-DR4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-XIAP, anti-NOXA, anti-BIM, anti-survivin, anti-Bid, anti-IRE1α, anti-phospho-IRE1α, anti-Bip, anti-GRP94, anti-ATF6, anti-eIF2α, anti-phospho-eIF2α, anti-CHOP, anti-cleaved PARP, anti-caspase-3, and anti-caspase-9 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). For the secondary antibodies, anti-mouse IgG horseradish peroxidase (HRP) and anti-rabbit IgG HRP were purchased from Cell Signaling Technology.

Kim J.L., Kim B.R., Kim D.Y., Jeong Y.A., Jeong S., Na Y.J., Park S.H., Yun H.K., Jo M.J., Kim B.G., Kim H.D., Kim D.H., Oh S.C., Lee S.I, & Lee D.H. (2019). Cannabidiol Enhances the Therapeutic Effects of TRAIL by Upregulating DR5 in Colorectal Cancer. Cancers, 11(5), 642.

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Evaluating Cellular Stress Markers in Breast Cancer Cell Lines

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MDA-MB-468 cells and MCF-7 were treated with the indicated concentrations of ChPL for 24 h, and Western blot analysis was performed according to the previously published procedure (Kawiak et al., 2012b (link)). The preparation of cytosolic and mitochondrial fractions for cytochrome c release evaluation was performed as previously described (Kawiak et al., 2012b (link)). The following specific primary antibodies were used: anti-β-actin (1:1,000) (Cell Signaling, Danvers, MA, USA), anti-Bcl-2, anti-Bak, anti-Bax, and anti-Mcl-1 (1:250) (Santa Cruz, Heidelberg, Germany), anti-ERK1/2, anti-MEK1/2, anti-p-ERK1/2, and anti-p-MEK1/2 (1:1,000) (Cell Signaling), anti-cytochrome c (1:5,000) (Abcam, UK), and anti-HSP60 (1:1,000) (Cell Signaling). Membranes were incubated with primary antibodies overnight at 4°C after which a 1-h incubation with HRP-conjugated secondary antibodies (1:2000) (Cell Signaling) was carried out. Protein levels were determined by chemiluminescence (ChemiDoc; Bio-Rad, Waltham, MA, USA) with a HRP substrate (Thermo Scientific, MA, USA).

Kawiak A., Domachowska A., Krolicka A., Smolarska M, & Lojkowska E. (2019). 3-Chloroplumbagin Induces Cell Death in Breast Cancer Cells Through MAPK-Mediated Mcl-1 Inhibition. Frontiers in Pharmacology, 10, 784.

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Apoptosis Pathway Antibody Panel

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Antibodies used include anti-Bid (Luo et al.^{12 (link)}), anti-Bim (Santa Cruz, Biotechnology, Inc, Dallas, TX, USA, Sc-11425, Calbiochem, San Diego, CA, USA, #202000), anti-Puma (Santa Cruz, Sc-28226, Pro-Sci, Inc, Poway, CA, USA, 3041), anti-Bad (Santa Cruz, Sc-8044), anti-Noxa (Santa Cruz, Sc-56169 Novus Biologicals, Littleton, CO, USA, IMG-349A), anti-Bax (Santa Cruz, Sc-493), anti-Bak (Santa Cruz, Sc-832, Cell Signaling Technology, Danvers, MA, USA, #3814), anti-Bcl-2 (Santa Cruz, Sc-509), anti-Bcl-xL (Santa Cruz, Sc-8392), anti-Mcl-1 (Santa Cruz, Sc-819), anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA, A5441), anti-PARP (Cell Signaling Technologies, #9524), anti-GFP (Santa Cruz, Sc-459), and anti-p53 (Santa Cruz, Sc-393). z-VAD(OMe)-FMK was purchased from MP Biomedicals (Santa Ana, CA, USA). Thapsigargin was purchased from Adipogen. Corp (San Diego, CA, USA) ABT-737 was purchased from Cayman Chemical (Ann Arbor, MI, USA). Human recombinant TRAIL was made as previously described.^{61 (link)}

Zhang J., Huang K., O'Neill K.L., Pang X, & Luo X. (2016). Bax/Bak activation in the absence of Bid, Bim, Puma, and p53. Cell Death & Disease, 7(6), e2266-.

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Apoptotic Pathway Regulation by Piceatannol and Gemcitabine

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Cells were treated with piceatannol and gemcitabine as single agents and as a combination and then harvested by centrifugation at 450 × g for 10 min at 4°C. Cells were washed with ice-cold PBS solution and scraped in lysis buffer. The lysates were centrifuged at 14,000 rpm for 30 min at 4°C, and the supernatant was collected. Equivalent amounts of protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Appropriate primary antibodies to anti-Bad, anti-Bak, anti-Bid, anti-Bcl-2, anti-Bcl-xl purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) and the antibodies against cytochrome c (Cyto-c) and GAPDH obtained from Cell Signaling Technology (Beverly, MA, USA) were used. Proteins were visualized with a HRP-conjugated goat anti-rabbit secondary antibody from Santa Cruz Biotechnology. Specific bands were detected using the enhanced chemiluminescence reagent (ECL; PerkinElmer Life Sciences, Inc., Waltham, MA, USA) on autoradiographic film.

Xu B, & Tao Z.Z. (2015). Piceatannol Enhances the Antitumor Efficacy of Gemcitabine in Human A549 Non-Small Cell Lung Cancer Cells. Oncology Research, 22(4), 213-217.

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Antioxidant Effects of Korean Red Ginseng

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Korean Red Ginseng powder was kindly provided by the Korea Ginseng Corporation (Daejeon, Korea). The KRG powders were dissolved in distilled water. N-acetyl-l-cysteine (NAC) and Anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase-3, anti-caspase-9, anti-cleaved PARP, anti-BIM, anti-Noxa, anti-survivin, anti-IRE1α, anti-phospho IRE1α, anti-GRP94, anti-eIF2α, anti-phospho eIF2α, anti-ATF6, anti-PERK, anti-phospho PERK, anti-Bip anti-XBP1s, anti-ATF4, anti-SOD2, anti-SOD1, anti-catalase, anti-NOX4, and anti-NOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bak, anti-BAX, anti-Bcl-2, anti-SOD3, and anti-CHOP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-mouse and Rabbit IgG HRP, the secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA).

Jeong Y.A., Kim B.R., Kim D.Y., Jeong S., Na Y.J., Kim J.L., Yun H.K., Kim B.G., Park S.H., Jo M.J., Lee S.I., Han B.C., Lee D.H, & Oh S.C. (2019). Korean Red Ginseng Extract Increases Apoptosis by Activation of the Noxa Pathway in Colorectal Cancer. Nutrients, 11(9), 2026.

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Bak Oligomerization and Conformational Change

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Cells were treated with agents and incubated with 1 mM BMH in 10% DMSO or DMSO alone for 30 min at 25°C. After centrifugation at 5000 g for 25 min at 4°C, the reaction was split into supernatant and pellet fractions. The pelleted material (10 mg total protein) was separated by SDS-PAGE and immunoblotted with anti-Bak antibody to detect Bak or Bax oligomerization [16 (link)]. Bak conformational change was performed as described [46 (link)]. Briefly, Cells were lysed in 1% CHAPS buffer, and 250 μg of protein was immunoprecipitated using anti-Bak (Ab-1; Merck), which only recognizes Bak that has undergone a conformation change. Immunoprecipitated protein was then subjected to immunoblot analysis by using anti-Bak (Santa Cruz) as primary antibodies.

Wang J., Guo W., Zhou H., Luo N., Nie C., Zhao X., Yuan Z., Liu X, & Wei Y. (2015). Mitochondrial p53 phosphorylation induces Bak-mediated and caspase-independent cell death. Oncotarget, 6(19), 17192-17205.

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Plumbagin and Tamoxifen Modulate Apoptosis Signaling

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MCF-7 and T47D cells were treated with plumbagin (0–2.5 μM) and/or with tamoxifen (1 μM) for 24 h and Western blot analysis was performed according to the previously published procedure. Specific primary antibodies: anti-GRP78, anti-Bik, anti-Bax, anti-Bak, anti-Bcl-2 (1:250) (Santa Cruz, Heidelberg, Germany), anti-β-actin (1:1000) (Cell Signaling, Germany), were incubated with membranes overnight at 4 °C. Membranes were further incubated at room temperature for 1 h with HRP-conjugated secondary antibodies (Cell Signaling, Germany) and proteins were detected by chemiluminescence (ChemiDoc, Bio-Rad) with a HRP substrate (Pierce).

Kawiak A., Domachowska A., Jaworska A, & Lojkowska E. (2017). Plumbagin sensitizes breast cancer cells to tamoxifen-induced cell death through GRP78 inhibition and Bik upregulation. Scientific Reports, 7, 43781.

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Western Blot Analysis of Protein Expression

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Western blotting was performed as previously described 21 (link).
The antibodies used were as follows: anti-Msi1 (1:1000 dilution, Abnova, Taipei, Taiwan), anti-PTEN (1:1000 dilution, Santa Cruz, CA, USA), anti-PI3K (1:1000 dilution, Santa Cruz, CA, USA), anti-p-AKT (1:1000 dilution, Cell Signaling Technology), anti-AKT1 (1:1000 dilution; Santa Cruz, CA, USA), anti-mTOR (1:1000 dilution, Santa Cruz, CA, USA), anti-BAK (1:500 dilution, Santa Cruz Biotechnology), anti-Bcl-2(1:1000 dilution, Santa Cruz, CA, USA), anti-GAPDH (1:1000 dilution, Santa Cruz, CA, USA), and the secondary antibody coupled to horseradish peroxidase (Goat aiti-Mouse:1:10000 dilution; Goat anti-Rabbit: 1:15000, Thermo Fisher Scientific Inc., New York, NY, USA). The proteins were visualized with an enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA) with the protein imprinting imaging system (Tanon 5200, China). GAPDH was used as the control and for quantification.

Liu X., Zhang Y., Zheng P, & Cui N. (2021). Msi1 inhibits cervical cancer cell apoptosis by downregulating BAK through AKT signaling. Journal of Cancer, 12(8), 2422-2429.

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Investigating Apoptosis Signaling in H9c2 Cells

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For Western blot analysis, H9c2 cells were washed with PBS and lysed in lysis buffer (50 mM Tris, pH 7.5; 0.5 M NaCl; 1.0 mM EDTA, pH 7.5; 10% glycerol; 1 mM basal medium Eagle; 1% Igepal-630; proteinase inhibitor cocktail tablet (Roche, Mannheim, Germany)) and the debris were pelleted by centrifuging at 12,000× g for 30 min. The supernatants were electrophoresed by sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto Immobilon™ PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked using 5% low-fat milk and 1% Tween 20 in PBS and the blotted proteins were probed with one of the following antibodies: anti-NFIL3, anti-Bax, anti-Bak, anti-Cyt C, anti-cleaved caspase-3, anti-α-tubulin, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IGF2, anti-IGF2R (Abcam, Cambridge, MA, USA), anti-IGF1R, anti-phospho-IGF1R, anti-PI3K, anti-phospho-Akt, and anti-phospho-PLCβ (Cell Signaling Technology, Beverly, MA, USA). Protein expression was detected with the ECL detection system (Millipore).

Chen W.K., Kuo W.W., Hsieh D.J., Chang H.N., Pai P.Y., Lin K.H., Pan L.F., Ho T.J., Viswanadha V.P, & Huang C.Y. (2015). CREB Negatively Regulates IGF2R Gene Expression and Downstream Pathways to Inhibit Hypoxia-Induced H9c2 Cardiomyoblast Cell Death. International Journal of Molecular Sciences, 16(11), 27921-27930.

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