Anti ask1

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-ASK1 is a laboratory reagent that detects the presence of ASK1 (Apoptosis Signal-regulating Kinase 1) protein. ASK1 is a serine/threonine protein kinase that plays a role in various cellular processes. This product can be used to identify and quantify ASK1 levels in biological samples through techniques such as Western blotting.

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Lab products found in correlation

Close spelling variants of this product

Anti-phospho-ASK1 (6 citations) Rabbit anti-ASK1 (3 citations) Anti-p-ASK1 (2 citations)

9 protocols using anti ask1

Western Blot Analysis of Signaling Proteins

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Equal amounts of cell lysates were separated by 12 % SDS-PAGE, and electrophoretically transferred to PVDF membrane. The membrane was blocked and probed with primary antibody (anti-ASK1, anti-phospho-ASK1, anti-p38, anti-phospho-p38, anti-ERK1/2, anti-phospho-ERK1/2, anti-ERK5, anti-phospho- ERK5, anti-JNK, anti-phospho-JNK from Cell Signaling Technology; anti-MEKK2, anti-NIS, anti-TSHR from Santa Cruz; anti-β-actin from Sigma) followed by HRP (horseradish peroxidase)-labeled goat anti-mouse IgG (Abcam) or HRP-labeled goat anti-rabbit IgG (Abcam). Chemiluminescence was used to analyze protein levels and β-actin was used as a protein loading control. Semi-quantitative analysis was conducted by using ImageJ 1.49v (National Institutes of Health, USA).

Shen C.T., Qiu Z.L., Song H.J., Wei W.J, & Luo Q.Y. (2016). miRNA-106a directly targeting RARB associates with the expression of Na+/I− symporter in thyroid cancer by regulating MAPK signaling pathway. Journal of Experimental & Clinical Cancer Research : CR, 35, 101.

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Western Blot Analysis of Immune Signaling

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The cells were lysed in radioimmunoprecipitation assay buffer (containing 1% protease and phosphatase inhibitor), and the total protein content was quantitatively measured using the bicinchoninic acid assay. Protein (35 μg) was separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a polyvinylidene fluoride membrane, blocked with 5% non-fat milk for 2 h, and incubated with the primary antibody overnight at 4°C. After washing the polyvinylidene fluoride membrane using tris-buffered saline with 0.1% tween, immunoreactivity was measured using rabbit HRP-conjugated secondary antibody (1:5000; Millipore), and the beta-actin level was measured as a control. The following primary antibodies were used: anti-SOCS3 (1:1000, 52113 s), anti-p38 (1:1000, 8690 s), and anti-ASK1 (1:1000, 8662 s) from Cell Signaling Technology (Beverly, MA, USA); anti-TLR2 (1:1000, ab16894) anti-LL37 (1:1000, ab180760) from Abcam (Cambridge, MA); anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Imaging was performed using a ChemiDoc^TM XRS+ system (Bio-Rad, USA) and an HRP substrate (Luminata; Millipore), and the bands on the western blots were scored by scanning and quantitatively analyzing grayscale values using ImageJ (National Institutes of Health, Bethesda, MD).

Liu Z., Zhang J., Jiang P., Yin Z., Liu Y., Liu Y., Wang X., Hu L., Xu Y, & Liu W. (2021). Paeoniflorin inhibits the macrophage-related rosacea-like inflammatory reaction through the suppressor of cytokine signaling 3-apoptosis signal-regulating kinase 1-p38 pathway. Medicine, 100(3), e23986.

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Immunoblotting Analysis of Autophagy Markers

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Protein sample preparation and Western immunoblotting were performed as previously described (Qin et al., 2011 (link); Pandey et al., 2017 (link)). Primary antibodies used in the immunoblotting analysis included anti-p-ULK1, anti-ULK1, anti-ASK1, anti-p-ASK1, anti-SAPK/JNK, anti-p-SAPK/JNK (Cell signaling); anti-AMPKα, anti-p-AMPKα, anti-Atg9a (Thermo Scientific); anti-LC3, anti-p-ASK1 and anti-GAPDH (Santa Cruz Biotech., Inc); anti-BAK, anti-BAX (EMD Millipore), anti-p-IRE1α (GeneTex, Inc), anti-IRE1α (Novus Biologicals). Dilution of primary antibodies was 1:1,000. Secondary antibody HRP anti-IgG (Sigma-Aldrich, USA, 1:1,000~5,000) was used in the immunoblotting analysis. Densitometry of blots was performed using the ImageJ software package (http://rsbweb.nih.gov/ij/). The relative expression levels of target proteins and the ratio of blot LC3-II/LC3-I at the indicated time points were calculated as previously described (Qin et al., 2011 (link)). All Westerns were performed in triplicate and representative images are shown.

Pandey A., Lin F., Cabello A.L., da Costa L.F., Feng X., Feng H.Q., Zhang M.Z., Iwawaki T., Rice-Ficht A., Ficht T.A., de Figueiredo P, & Qin Q.M. (2018). Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis. Frontiers in Cellular and Infection Microbiology, 8, 103.

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Characterization of CFTR Regulatory Pathways

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Thapsigargin (Sigma-Aldrich, T9033), STF-083010 (TOCRIS, 4509), APY29 (TOCRIS, 4865), MSC 2032964A (TOCRIS, 5641), VX-809 (Selleckchem, Chemical Abstracts Service registry no. 936727-05-8), VX-770 (Selleckchem, CAS registry no. 873054-44-5), and cycloheximide (Sigma-Aldrich, C4859) were purchased commercially. CSTMP was custom-synthesized by Cayman Chemical (Ann Arbor, MI, USA) (CAS registry no. 1000672-89-8).
The following antibodies were acquired commercially: anti-CFTR M3A7 (Millipore, Billerica, MA, USA), anti-IRE1α (Cell Signaling Technology, 3294), anti–phospho-S724 IRE1α (Abcam, ab48187), anti-XBP1 (Abcam, ab198999), anti-ASK1 (Cell Signaling Technology, 3762), anti–phospho-Thr⁸⁴⁵ ASK1 (Cell Signaling Technology, 3765), anti-BiP (Cell Signaling Technology, 3177), anti-CHOP (Cell Signaling Technology, 2895), anti-pro/p17–caspase 3, anti–cleaved PARP1 (Abcam, ab136812), anti–Na- and K-dependent ATPase (Na,K-ATPase) α1 (Cell Signaling Technology, 23565), anti-HA (Cell Signaling Technology, 2367), anti-Myc (Cell Signaling Technology, 2276), anti–aldolase A (Abcam, ab78339) anti–β-actin (Santa Cruz Biotechnology, sc47778), and anti-pendrin (Santa Cruz Biotechnology, sc23779). The anti-R4 polyclonal antibody was raised against peptides corresponding to amino acids 1458 to 1471 of human CFTR, as described previously (12 (link)).

Park H., Shin D.H., Sim J.R., Aum S, & Lee M.G. (2020). IRE1α kinase–mediated unconventional protein secretion rescues misfolded CFTR and pendrin. Science Advances, 6(8), eaax9914.

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Gallic Acid-Mediated Gold Nanoparticle Synthesis

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Gallic acid, hydrogen tetrachloroaurate (HAuCl₄), and collagen I were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). The CellTiter 96^® aqueous one-solution proliferation assay kit was obtained from Promega Corporation Inc. (Madison, WI, USA). H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-MMP-1 and anti-MMP-3 were purchased from Abcam (San Francisco, CA, USA). Anti-β-actin and peroxidase conjugated secondary antibodies were obtained from EMD Millipore Inc. (Burlington, MA, USA). Anti-phospho-NFκB p65 (Ser536), anti-NFκB p65, anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-ASK1, anti-ASK1, anti-phospho-c-Jun, anti-c-Jun, anti-phospho-c-Fos, anti-c-Fos, anti-phospho-ATF-2, and anti-ATF-2 antibodies were the products of Cell Signaling Technology (Beverly, MA, USA). Cell culture supplies were purchased from GIBCO/Life Technologies Inc. (Carlsbad, CA, USA).

Wu Y.Z., Tsai Y.Y., Chang L.S, & Chen Y.J. (2021). Evaluation of Gallic Acid-Coated Gold Nanoparticles as an Anti-Aging Ingredient. Pharmaceuticals, 14(11), 1071.

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Recombinant Protein Immunoblotting Analysis

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Recombinant MKK6 was obtained from Millipore (Watford, UK). Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐c‐myc came from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti‐GST and anti‐maltose‐binding protein (MBP) from MBL (Nagoya, Japan), Anti‐ROR1 and anti‐goat IgG from R&D Systems (Minneapolis, MN, USA), anti‐JNK and anti‐phospho‐JNK (T183/Y185) from BD Biosciences (Bedford, MA, USA), and anti‐α‐tubulin from Sigma (St. Louis, MO, USA).

Ida L., Yamaguchi T., Yanagisawa K., Kajino T., Shimada Y., Suzuki M, & Takahashi T. (2016). Receptor tyrosine kinase‐like orphan receptor 1, a target of NKX2‐1/TTF‐1 lineage‐survival oncogene, inhibits apoptosis signal‐regulating kinase 1‐mediated pro‐apoptotic signaling in lung adenocarcinoma. Cancer Science, 107(2), 155-161.

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Protein Extraction and Western Blot Analysis

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Preparation of total protein extracts from mice liver or cells was performed following the procedure described previously [36 (link)]. The extracted proteins and immunoprecipitates were separated by SDS-PAGE 10% or 12% polyacrylamide gel and then electrically transferred to a PVDF membrane. After blocking with 5% (w/v) BSA in TBST at room temperature for 1 h, the membranes were then incubated with an appropriate specific primary antibody (Anti-JNK1/2, anti-phospho JNK1/2, anti- MKK4, anti-phospho MKK4, anti-ASK1, anti-phospho ASK1, anti-Ubiquitinin, Cell Signaling Technology, Boston; Anti-mono and dimethyl Arginine, Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibody (1:15,000; Sunshine Biotechnology) and detected by enhanced chemical luminescence kit (Thermo scientific, Hudson, NH, USA). The quantification of the bands was performed by the Gel Analysis V2.02 Software (Clin Science Instruments, Shanghai, China).

Yang X., Zhan Y., Sun Q., Xu X., Kong Y, & Zhang J. (2016). Adenosine 5′-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation. Oncotarget, 8(4), 6273-6282.

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ASK1 Immunoprecipitation in Platelets

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Immunoprecipitation was performed as previously described [16 (link)]. Briefly, washed human platelet suspension (4 x 10⁸ platelets/mL) was stimulated with thrombin (1U/mL) for 3 min at room temperature. Platelet suspensions were lysed by the addition of an equal volume of ice-cold 2X lysis buffer to reach a final concentration of (1% Triton X-100, 150mM NaCl, 50 mM Tris-HCl pH 7.5, 10μg/mL leupeptin, 10μg/mL aprotinin, 1mM NaF, 1mM sodium orthovanadate, and 1mM PMSF). Pre-cleared lysates were incubated with anti-ASK1 [Santa Cruz Biotechnology, 1:100] for IP of human ASK1, or normal rabbit IgG [Santa Cruz Biotechnology, 1:100] and the immunoprecipitation was performed as described [16 (link)]. Precipitate was western blotted with anti-ASK1, anti-CIB1, anti-TRAF6, and anti-Trx1 antibodies.
IP of mouse Ask1 followed the above protocol with the following modifications. Briefly, washed mouse platelet suspension (4 x 10⁸ platelets/mL) was stimulated with thrombin (1U/mL) for 1 min at room temperature before being lysed with ice-cold 2X lysis buffer. Lysates were then incubated with anti-ASK1 [Cell Signaling, 0.23 μg/mL] for IP of mouse Ask1, or normal rabbit IgG [Santa Cruz Biotechnology, 0.23μg/mL] as control and the immunoprecipitation was performed as described [16 (link)]. Precipitate was western blotted with anti-ASK1 and anti-Traf6 antibodies.

Patel P., Naik M.U., Golla K., Shaik N.F, & Naik U.P. (2019). Calcium-induced dissociation of CIB1 from ASK1 regulates agonist-induced activation of the p38 MAPK pathway in platelets. The Biochemical journal, 476(19), 2835-2850.

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Colorectal Cancer Cell Lines: Characterization and Manipulation

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The human colorectal cancer cell line SW480 and HT-29 (both from epithelial colorectal adenocarcinoma) were purchased from the American Type Culture Collection (Rockville, MD). The SW480/Vector, SW480/NNMT-1, SW480/NNMT-2 and HT-29/NC, HT-29/NNMT shRNA 1# and HT-29/NNMT shRNA 2 # cells were constructed by the method in our previous study [15 (link)]. SW480 cells were cultured in RPMI-1640 medium and HT-29 cells were cultured in McCoy's 5A medium. All media were supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin, and cells were maintained at 37°C in a humidified 5% CO₂ incubator.
The following antibodies were obtained from Cell Signaling Technology (Beverly, Massachusetts, USA): anti-ASK1, anti-phospho-ASK1 (Thr 845), anti-caspase-3, anti-cleaved caspase-3, anti-caspase-9, anti-cleaved caspase-9, anti-p38, anti-phospho-p38, anti-GAPDH. The mouse anti-NNMT monoclonal antibody 1E7 was prepared through the hybridoma technique as previously described [19 (link)]. Goat anti-mouse and goat anti-rabbit HRP-conjugated antibodies were obtained from Zhongshan Goldenbridge Biotechnology Co. (Beijing, China).

Xie X., Liu H., Wang Y., Zhou Y., Yu H., Li G., Ruan Z., Li F., Wang X, & Zhang J. (2016). Nicotinamide N-methyltransferase enhances resistance to 5-fluorouracil in colorectal cancer cells through inhibition of the ASK1-p38 MAPK pathway. Oncotarget, 7(29), 45837-45848.

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