Hexokinase 2

Manufactured by Cell Signaling Technology

Sourced in United States

Hexokinase II is a lab equipment product from Cell Signaling Technology. It is an enzyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate, which is the first step in glycolysis.

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Lab products found in correlation

Close spelling variants of this product

Anti-Hexokinase 2 (12 citations) Hexokinase 2 (HK2) (5 citations) Hexokinase II (HKII) (4 citations) Rabbit anti-Hexokinase 2 (2 citations)

37 protocols using hexokinase 2

Metabolic Reprogramming in Lung Cancer

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The normal human bronchial epithelioid cell (HBE) and the non-small-cell lung cancer cell lines including NCI-H1650, NCI-H1299, A549, NCI-H460, HCC4006, NCI-H1975, and NCI-H358 were obtained from ATCC and cultured with recommended culture medium. The primary antibodies for western blotting including anti-TRAF6 (#8028), hexokinase-1 (#2024), hexokinase-2 (#2867), GLUT1 (#12939), PKM2 (#4053), LDHA (#35 82), VDAC-1 (#4661), phosphor-Akt (#4060), Akt (#8596), phosphor-S6 (#4858), and ubiquitin (#58395) as well as the secondary anti-rabbit IgG HRP (#7074) were products of Cell Signaling Technology Inc. (Danvers, MA). β-Actin (A5316) was obtained from Sigma-Aldrich. In immunohistochemistry staining, the primary antibodies against hexokinase-2 (ab227198) and Ki67 (ab15580) were products of Abcam. TRAF6 shRNA#1 (TRCN0000007350) and shRNA#2 (TRCN0000007351) were purchased from the Sigma Mission shRNA library. The constitutively active Akt (CA-Akt) plasmid (Cat. #10841) and pLKO.1 GFP shRNA (Cat. #30323) were purchased from Addgene (Cambridge, MA, USA). Recombinant human insulin-like growth factor 1 (IGF-1) was a product of R&D (Cat. 291-G1-200). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA).

Feng L., Feng S., Nie Z., Deng Y., Xuan Y., Chen X., Lu Y., Liang L, & Chen Y. (2021). TRAF6 Promoted Tumor Glycolysis in Non-Small-Cell Lung Cancer by Activating the Akt-HIFα Pathway. BioMed Research International, 2021, 3431245.

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Immunoblotting and Antibody Detection

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Immunoblotting was performed as described previously (Jacobs et al., 2008). Primary antibodies were followed by mouse- or rabbit-conjugated horseradish peroxidase (HRP). HRP-conjugated antibodies (anti-mouse or anti-rabbit IgG HRP conjugate, Promega) were detected by enhanced chemiluminescence detection (Thermofisher). This included the following antibodies: ACC (3662, Cell Signaling), phospho-ACC (3661, Cell Signaling), pan-AMPKα (2532, Cell Signaling), phospho-AMPKα (2535, Cell Signaling), 4EBP1 (9644, Cell Signaling), phospho-4EBP1 (2855, Cell Signaling), c-myc (9402, Cell Signaling), phospho-mTOR (5536, Cell Signaling), Activated Notch (ab8925, Abcam), RAPTOR (2280, Cell Signaling), phospho-RAPTOR (2083, Cell Signaling), S6 (2217, Cell Signaling), phospho-p70 S6K (9204, Cell Signaling), p70 S6K (2708, Cell Signaling), phosphor-TSC2 (5584, Cell Signaling), TSC2 (3612, Cell Signaling). Alternatively, primary antibodies were followed by fluorescently labeled anti-mouse or rabbit antibodies (LiCor) and imaged using the Odyssey infrared imaging system (LiCor). This included the following antibodies: Glut1 (ab652, Abcam), hexokinase 2 (2867, Cell Signaling), hexokinase 1 (ab104835, Abcam), cytochrome C (556433, BD Biosciences), β-actin (A5441, Sigma), phospho-S6 (4858, Cell Signaling). Western blots were quantified using ImageJ software.

Kishton R.J., Barnes C.E., Nichols A.G., Cohen S., Gerriets V.A., Siska P.J., Macintyre A.N., Goraksha-Hicks P., de Cubas A.A., Liu T., Warmoes M.O., Abel E.D., Yeoh A.E., Gershon T.R., Rathmell W.K., Richards K.L., Locasale J.W, & Rathmell J.C. (2016). AMPK is essential to balance glycolysis and mitochondrial metabolism to control T-ALL cell stress and survival. Cell metabolism, 23(4), 649-662.

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Immunoblotting Antibody Specifications

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Antibodies for immunoblotting such as p‐AMPKα (#2535), AMPKα (#5831), PDK1 (#3820), HIF1α (#3716), hexokinase 2 (#2867), LDHA (#3582), PFKP (#8164), PGAM1 (#12098), PDH (#3205), p‐Threonine/Tyrosine (#9381), p‐S6K (#9234), S6K (#2708), p‐ERK (#4370), ERK (#4695), p‐4E‐BP1 (Thr37/46, #2855), p‐4E‐BP1 (Ser65, #9451), 4E‐BP1 (#9644), mouse antirabbit IgG mAb (#5127), and antirabbit IgG (#7074) were from Cell Signaling Technology (Danvers, MA, USA). HIF1α (hydroxyl P564) (ab72777) and PHD3 (ab30782) were purchased from Abcam (Cambridge, MA, USA). HIF1α (hydroxyl P402) (07‐1585) was purchased from Merck (Darmstadt, Germany). GAPDH (sc‐25778) was purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). Actin (GTX109639) was purchased from GeneTex International (Hsinchu, Taiwan). Antibodies for immunoprecipitation such as HIF1α (#36169), rabbit mAb IgG (#3900), and p‐AMPKα (#2535) were purchased from Cell Signaling Technology. AMPKα1 (sc‐398861) was purchased from Santa Cruz Biotechnology. Antibodies for IHC such as p‐AMPKα (GTX52341) and HIF1α (GTX127309) were purchased from GeneTex International.

Tseng H., Zeng Y., Lin Y.J., Huang J., Lin C., Lee M., Yang F., Fang T., Mar A, & Su J. (2022). A novel AMPK activator shows therapeutic potential in hepatocellular carcinoma by suppressing HIF1α‐mediated aerobic glycolysis. Molecular Oncology, 16(11), 2274-2294.

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Antibody Validation for Immunoblot Analysis

Cited 5 times

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Immunoblots were conducted as previously described^{18 (link)}. The following antibodies were used: FLT3 (Cell Signaling Technology Cat# 3462, RRID:AB_2107052, validated through overexpression (in this work) and knockdown studies^{45 (link)}); JAK2 (Cell Signaling Technology Cat# 3230, RRID:AB_2128522, validated through overexpression (in this work) and knockdown studies^{46 (link)}); GLUT1 (Millipore Cat# 07–1401, RRID:AB_1587074, validated through overexpression^{18 (link)} and knockdown studies^{47 (link)}); GLUT3 (Abcam Cat# ab15311, RRID:AB_301846, validated through overexpression studies^{18 (link)}); Hexokinase 1 (Cell Signaling Technology Cat# 2024, RRID:AB_2116996, validated through overexpression (in this work) and knockout studies^{48 (link)}); Hexokinase 2 (Cell Signaling Technology Cat# 2867, RRID:AB_2232946, validated through overexpression (in this work) and knockout studies^{48 (link)}); β-actin (Cell Signaling Technology Cat# 4970, RRID:AB_2223172, used as a loading control in various studies^{49 (link)–51 (link)}).

Ghezzi C., Chen B.Y., Damoiseaux R, & Clark P.M. (2023). Pacritinib inhibits glucose consumption in squamous cell lung cancer cells by targeting FLT3. Scientific Reports, 13, 1442.

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Western Blot Analysis of Prostate Samples

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Cells were lysed with TEB150 buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 5 mM EGTA pH 8.0, 1 mM dithiothreitol, 0.5% Triton X–100, 10% glycerol, 1 mM Na₃VO₄, 1 μM microcystin–LR and protease/phosphatase inhibitor cocktail). Lysates from mouse prostates were prepared by homogenizing mouse prostates in modified RIPA buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 5 mM EDTA and protease/phosphatase inhibitor cocktail). Insoluble material was removed by centrifugation. Proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% non-fat milk in phosphate-buffered saline and then probed with the appropriate primary antibody in 5% non-fat milk overnight at 4 °C [AMPKα, phospho-AMPKα (Thr172), Bax, cleaved PARP, cleaved caspase-3, Hexokinase 2, ATG5, ATG7, Beclin 1, ULK1, and LC3-II from Cell Signaling; β Tubulin (G-8) and Bcl-2 (N-19) from Santa Cruz Biotechnology; AR for IHC from Millipore and AR for Western blot from Santa Cruz; and LC3 (M115–3) from MBL International Corporation]. Subsequently, membranes were washed in phosphate-buffered saline with 0.3% Tween 20, and incubated with secondary antibody conjugated to horseradish peroxidase. Proteins were visualized by enhanced chemiluminescence.

Wang L., Wang J., Xiong H., Wu F., Lan T., Zhang Y., Guo X., Wang H., Saleem M., Jiang C., Lu J, & Deng Y. (2016). Co-targeting hexokinase 2-mediated Warburg effect and ULK1-dependent autophagy suppresses tumor growth of PTEN- and TP53-deficiency-driven castration-resistant prostate cancer. EBioMedicine, 7, 50-61.

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Molecular Mechanisms of Cancer Metabolism

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CANA, MG132, LW-6 and cycloheximide (CHX) were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Cobaltous chloride (CoCl₂) was obtained from Sigma-Aldrich (Chicago, NJ, USA). The specific primary antibodies used for Western blotting analysis against protein kinase B (AKT) (1:1000), p-AKT (1:1000), mTOR (1:1000), p-mTOR (1:1000), HIF-1α (1:1000), fibronectin (FN-1) (1:1000), lactate dehydrogenase A (LDHA) (1:1000), glucosetransporter1 (GLUT1) (1:1000), hexokinase 2 (HK2) (1:1000), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) (1:1000), β-catenin (1:1000), E-cadherin, snail, zonula occludens-1 (ZO-1) (1:1000) and talin (1:1000) were purchased from Cell Signaling Technology (Boston, MA, USA), while actin (1:50,000) was purchased from Sigma-Aldrich^® (Darmstadt, Germany), and vascular endothelial growth factor A (VEGFA) (1:500), P70S6K (1:2000), and p-P70S6K (1:2000) were purchased from Abclonal (Wuhan, China). The secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Luo J., Sun P., Zhang X., Lin G., Xin Q., Niu Y., Chen Y., Xu N., Zhang Y, & Xie W. (2021). Canagliflozin Modulates Hypoxia-Induced Metastasis, Angiogenesis and Glycolysis by Decreasing HIF-1α Protein Synthesis via AKT/mTOR Pathway. International Journal of Molecular Sciences, 22(24), 13336.

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Western Blot Analysis of Glycolytic Enzymes

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Cells were lysed in modified RIPA buffer (50 mM Tris–HCl (pH 7.5), 150 NaCl, 10 mM β‐glycerophosphate, 1% NP‐40, 0.25% sodium deoxycholate, 10 mM sodium pyrophosphate, 30 mM sodium fluoride, 1 mM EDTA, 1 mM vanadate, 20 μg/ml aprotinin, 20 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Whole‐cell lysates were resolved by SDS–PAGE on 4–15% gradient gels and blotted onto nitrocellulose membranes (Bio‐Rad). Membranes were blocked overnight and then incubated sequentially with primary and either HRP‐conjugated (Pierce) or IRDye‐conjugated secondary antibodies (Li‐Cor). Blots were imaged using the Odyssey Infrared Imaging System (Li‐Cor). Protein levels were quantitated using ImageJ (http://imagej.nih.gov/ij/). Primary antibodies used for Western blot analysis included hexokinase 1 (2024, Cell Signaling Technology), hexokinase 2 (2867, Cell Signaling Technology), p53 (NB200‐103, Novus Biologicals), and enolase 2 (8171, Cell Signaling Technology).

Graham N.A., Minasyan A., Lomova A., Cass A., Balanis N.G., Friedman M., Chan S., Zhao S., Delgado A., Go J., Beck L., Hurtz C., Ng C., Qiao R., ten Hoeve J., Palaskas N., Wu H., Müschen M., Multani A.S., Port E., Larson S.M., Schultz N., Braas D., Christofk H.R., Mellinghoff I.K, & Graeber T.G. (2017). Recurrent patterns of DNA copy number alterations in tumors reflect metabolic selection pressures. Molecular Systems Biology, 13(2), 914.

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Investigating Cellular Signaling Pathways

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Cell lysates from H460, A549, H1975, and HCC827 cells were prepared 24 h after treatment with DMSO, Milciclib (1 or 10 μM), THZ1 (10 μM), or LDC4297 (10 μM); 16 h after treatment with 1× PBS or Deferoxamine (100 μM); or 72 h after transfection with shRNA or an overexpression plasmid. Uncropped immunoblots are presented in Supplementary Fig. 29.
The following antibodies were used: HIF-1α (Novus Biologicals; NB100-105; 1:1000 dilution), GLUT1 (Millipore Sigma; 07-1401; 1:500 dilution), GLUT3 (Abcam; ab15311; 1:1000 dilution), Hexokinase 1 (Cell Signaling; C35C4; 1:1000 dilution), Hexokinase 2 (Cell Signaling; C64G5; 1:1000 dilution), CDK2 (Cell Signaling; 78B2; 1:1000 dilution), CDK4 (Cell Signaling; D9G3E; 1:1000 dilution), CDK7 (Cell Signaling; MO1; 1:1000 dilution), TRKA (Cell Signaling; 12G8; 1:1000 dilution), PIK3CA (Cell Signaling; C73F8; 1:1000 dilution), PTEN (Cell Signaling; 9559; 1:1000 dilution), Phospho-T170 CDK7 (Abcam; ab155976; 1:1000 dilution), Rpb1 NTD (Cell Signaling; D8L4Y; 1:1000 dilution), Phospho-S5 Rpb1 (Cell Signaling; D9N5I; 1:1000 dilution), PKCι (BD Biosciences; Clone 23; 1:1000 dilution), TXNIP (Cell Signaling; D5F3E; 1:1000 dilution), and Beta Actin (Imgenex; IMG-5142; 1:1000 dilution). Immunoblots were analyzed using an Odyssey Imaging System (LI-COR).

Ghezzi C., Wong A., Chen B.Y., Ribalet B., Damoiseaux R, & Clark P.M. (2019). A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells. Nature Communications, 10, 5444.

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Comprehensive Inhibitor Screening for Cancer

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Compounds utilized in our studies, including the small molecule inhibitors panobinostat, marizomib, 2‐DG, IACS‐010759, lonidamine, antimycin A, FCCP, and Gboxin, were purchased from Selleck Chemicals (Houston, TX, USA). Fluorescent probes were purchased from Invitrogen/Molecular Probes (Eugene, OR, USA) unless otherwise stated. The following antibodies were used: BiP (#3177), cleaved caspase 3 (#9664), cleaved caspase 7 (#8438), caspase 8 (#9746), Enolase‐2 (#24330), phospho‐H2AX (#80312), GAPDH (#2118), Hexokinase‐2 (#2867), PGC‐1α (#2178), RAD51 (#8875), and β‐Actin (#4970); all were from Cell Signaling Technology Inc., (Beverly, MA, USA).

Jane E.P., Reslink M.C., Gatesman T.A., Halbert M.E., Miller T.A., Golbourn B.J., Casillo S.M., Mullett S.J., Wendell S.G., Obodo U., Mohanakrishnan D., Dange R., Michealraj A., Brenner C., Agnihotri S., Premkumar D.R, & Pollack I.F. (2023). Targeting mitochondrial energetics reverses panobinostat‐ and marizomib‐induced resistance in pediatric and adult high‐grade gliomas. Molecular Oncology, 17(9), 1821-1843.

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HIF-1α Regulation of Glycolysis

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All chemicals were purchased from Sigma-Aldrich, unless indicated otherwise. Rabbit polyclonal antibody against HIF-1α was obtained from (Novus Biologicals, Littleton, CO, USA, #NB100–449), while phosphorylated mTOR S2448, β-actin, α-Tubulin, as well as Glycolysis Antibody Sampler Kit to detect Pfkp, Pkm2, Pkm 1/2, Hexokinase 1, Hexokinase 2 and Pdh were all obtained from (Cell Signaling Technology, Beverly, MA, USA, #2971, #4970, #2148, #8337 respectively). Secondary antibodies consisted of horseradish peroxidase conjugated goat anti-rabbit IgG (Agilent’s Dako, Glostrup, Denmark). Torin 1, LY294002 and Forskolin were purchased from (ApexBio, Houston, TX, USA). ON-TARGETplus Rat Hif1a siRNA – SMARTpool (siHif1a) and Non-targeting Pool siRNA (siControl) were purchased from (Dharmacon, Lafayette, CO, USA - L-091718-02 and D-001810-10-05, respectively).

Carlessi R., Chen Y., Rowlands J., Cruzat V.F., Keane K.N., Egan L., Mamotte C., Stokes R., Gunton J.E., Bittencourt P.I, & Newsholme P. (2017). GLP-1 receptor signalling promotes β-cell glucose metabolism via mTOR-dependent HIF-1α activation. Scientific Reports, 7, 2661.

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