Hexokinase 2

Manufactured by Abcam

Sourced in United States

Hexokinase II is an enzyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate, which is the first step in glucose metabolism. It is a key regulator of glycolysis and plays an important role in cellular energy production.

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Hexokinase 2 (HK2; (5 citations) Anti-hexokinase 2 (anti-HK2) (2 citations)

8 protocols using hexokinase 2

Protein Expression Analysis by Western Blot

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Cell protein extraction and Western blot analyses were performed, as previously described [4 (link)]. Nitrocellulose membranes were immunoblotted with the following primary antibodies: β-actin, KLF4 (Cell Signaling, Danvers, MA, USA), hexokinase2 (HK2), monocarboxylate transporter 4 (MCT4), pyruvate kinase M2 (PKM2), GLUT1 (Abcam, Cambridge, UK), microtubule-associated proteins 1A/1B light chain 3B (LC3), and caspase-3 (Santa Cruz Biotechnology, Dallas, TX, USA). The blots were then incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA) for 1 h at room temperature. The antibody–antigen complex was visualized using ImageLab (Bio-Rad, Hercules, CA, USA). ImageLab was also used to quantify the Western band intensity (N = 4).

Blum A., Mostow K., Jackett K., Kelty E., Dakpa T., Ryan C, & Hagos E. (2021). KLF4 Regulates Metabolic Homeostasis in Response to Stress. Cells, 10(4), 830.

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Mitochondrial Protein Expression Analysis

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ADSCs were lysed using RIPA lysis buffer, and the total protein concentration was measured with a BCA protein assay kit (all from ThermoFisher Scientific). The transferred protein on the PVDF membrane (Millipore) was blocked with 5% non-fat milk for 1 h, followed by incubation with primary antibodies overnight at 4 °C. After washing with TBST, the PVDF membrane was incubated with the corresponding HRP-conjugated second antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. The primary antibodies used were: β-Actin (1:1,000), Tom20 (1:1000), Hexokinase 1 (HK1, 1:1000); Hexokinase 2 (HK2, 1:1000); Phosphofructokinase (PFK, 1:1000); Lactate dehydrogenase A (LDHA, 1:1000); Citrate Synthase (CS, 1:1000); Dihydrolipoamide succinyltransferase (DLST, 1:1000), Succinate dehydrogenase A (SDHA, 1:1000), and Fumarase (FH, 1:1000) (all obtained from Abcam). Mitochondrial fission factor (MFF, 1:1000); Mitofusin 2 (MFN2, 1:1000); Opticatrophy protein 1 (OPA1, 1:1000) (all obtained from Proteintech).

Wang B., Zhang G., Hu Y., Mohsin A., Chen Z., Hao W., Li Z., Gao W.Q., Guo M, & Xu H. (2024). Uncovering impaired mitochondrial and lysosomal function in adipose-derived stem cells from obese individuals with altered biological activity. Stem Cell Research & Therapy, 15, 12.

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Western Blot Analysis of Cellular Metabolism

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UMSCs were lysed in RIPA lysis buffer (Thermo Fisher Scientific), and total protein concentration was measured with BCA protein assay kit (Thermo Fisher Scientific). The PVDF membrane (Invitrogen) transferred protein was blocked with 5% non-fat milk and then incubated with primary antibodies overnight at 4 ℃. After washing with TBST for three times, the PVDF membrane was incubated with corresponding HRP-conjugated second antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. The primary antibody information is as follows, Bcl-2 (1:1000, abclonal), cleaved Caspase-3 (1:1000, abclonal), glutathione peroxidase 4 (GPX4, 1:1000); hexokinase 1 (HK1, 1:1000); hexokinase 2 (HK2, 1:1000); phosphofructokinase (PFK, 1:1000); lactate dehydrogenase A (LDHA, 1:1000); citrate synthase (CS, 1:1000); aconitase 2 (ACO2, 1:1000); fumarase (FH, 1:1000); succinate dehydrogenase A (SDHA, 1:1000); all were obtained from Abcam.

Zhang G., Wang Y., Lin J., Wang B., Mohsin A., Cheng Z., Hao W., Gao W.Q., Xu H, & Guo M. (2022). Biological activity reduction and mitochondrial and lysosomal dysfunction of mesenchymal stem cells aging in vitro. Stem Cell Research & Therapy, 13, 411.

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Immunohistochemical Profiling of Tumor Samples

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Harvested tissues were immediately fixed in 10% formalin overnight and embedded in paraffin. IHC was performed as described previously^{62 (link)}. Briefly, endogenous peroxidases were inactivated by 3% hydrogen peroxide. Non-specific signals were blocked using 3% BSA, 10% goat serum in 0.1% Triton X-100. Tumor samples were stained with the following primary antibodies: IL2Rγ (Abcam ab180698, Bioss bs-2545R); Ki67 (Vector Laboratory, VP-RM04); IL4R (Bioss bs2458R); cMyc (Abcam ab32072); Hexokinase II (Abcam ab209847); Gata-3 (CST 5852); LDHA (CST 3582); IL13rα1 (Abcam ab79277); Jak1 (CST 3344); pSTAT1-Y701 (CST 9167); pSTAT3-Y705 (CST 9145); Stat5 (CST 94205); Stat1 (CST 9172); IL4 (Abcam ab9622); IL13 (Abcam ab106732); CD45 (Abcam ab10558), CD4 (Abcam ab183685) and F4/80 (Abcam ab6640). After overnight incubation, the slides were washed and incubated with secondary antibody (HRP-polymers, Biocare Medical) for 30 min at room temperature. The slides were washed three times and stained with DAB substrate (ThermoFisher Scientific). The slides were then counterstained with haematoxylin and mounted with mounting medium. For clinical samples, staining intensity of tissue sections was scored in a ‘blinded’ manner by two independent pathologists.
Immunofluorescence slides were imaged with an Olympus Microscope and quantified with ImageJ.

Dey P., Li J., Zhang J., Chaurasiya S., Strom A., Wang H., Liao W.T., Cavallaro F., Denz P., Bernard V., Yen E.Y., Genovese G., Gulhati P., Liu J., Chakravarti D., Deng P., Zhang T., Carbone F., Chang Q., Ying H., Shang X., Spring D.J., Ghosh B., Putluri N., Maitra A., Wang Y.A, & DePinho R.A. (2020). Oncogenic Kras driven metabolic reprogramming in pancreas cancer cells utilizes cytokines from the tumor microenvironment. Cancer discovery, 10(4), 608-625.

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Molecular Mechanisms of EMT and Metabolism

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Recombinant human TNFα and TGFβ were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies used are listed as follows: IkB, phospho-Smad2/3, total-Smad2/3, Snail, vimentin, lactate dehydrogenase-A, fatty acid synthase, stearoyl-CoA desaturase-1, phospho-acetyl-CoA carboxylase, total-acetyl-CoA carboxylase (Cell signaling, MA, USA); beta-actin, E-cadherin, N-cadherin (Santa Cruz, TX, USA); hexokinase II, pyruvate kinase isoform 2, fructose-bisphophotase-2, total OXPHOS Human WB Antibody Cocktail (Abcam, Cambridge, UK); Zeb1 (ProSci, CA, USA); and fructose-bisphophotase-1 (Abgent, Wuxi, China). All other reagents were from Sigma-Aldrich (MO, USA) unless stated otherwise.

Liu M., Quek L.E., Sultani G, & Turner N. (2016). Epithelial-mesenchymal transition induction is associated with augmented glucose uptake and lactate production in pancreatic ductal adenocarcinoma. Cancer & Metabolism, 4, 19.

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Protein Expression Analysis by Western Blot

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Total proteins were extracted from cells, and western blotting was performed as previously reported^{33 (link)}. The primary antibodies used were: PCNA (Cell Signaling Technology, Danvers MA, USA; #13110; dilution 1:5000); Hexokinase II (EPR20839; 1:500), HIF1α(1:1000), GLUT1 (1:2000), N cadherin (5D5; 1:1000), and Snail/Slug (1:1000), all procured from Abcam (Cambridge, UK); and β-actin-HRP (Santa Cruz Biotechnology, Dalla TX, USA; C4; dilution 1:1000). Western blotting experiments from biological replicates showed similar expression data, attesting to the reproducibility of the results. For band quantification, images were analyzed using Image Lab software (Bio-Rad, Hercules, California, USA). For band quantification, images were analyzed using IMT i-Solution software Martin Microscope Company, Easley, USA).

Jo H., Lee J., Jeon J., Kim S.Y., Chung J.I., Ko H.Y., Lee M, & Yun M. (2020). The critical role of glucose deprivation in epithelial-mesenchymal transition in hepatocellular carcinoma under hypoxia. Scientific Reports, 10, 1538.

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Comprehensive Protein Profiling Technique

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Media were removed and the cells were washed twice in ice-cold phosphate-buffered saline (PBS), scraped and collected as pellets after centrifugation at 1700 g for 5 min. The pelleted cells were incubated in RIPA buffer with proteinase and phosphatase inhibitors for 15 min. Lysates were then collected and centrifuged at 208,000 × g for 15 min at 4°C. Protein concentrations were measured using the DC Protein Assay Kit (Biorad, Cat. No. 5000111). SDS–PAGE and immunoblotting were performed as described previously in pre-cast bis-Tris 4–20% gradient gels (Invitrogen)^{64 (link)}. The following antibodies were used: IL2rγ (Abcam ab180698, bioss bs-2545R); IL13ra1 (Abcam ab79277); pAKT-S473 (CST 9271); Stat1 (CST 9172); Stat3 (CST 4904); pStat3-S727 (CST 9134); pStat2-Y690 (4441); pStat5-Y705 (CST 9145); Stat6 (abcam ab28829); Stat6 (CST); pStat6-Y641 (CST 56554); pStat5-Y694 (CST 4322); pStat5 (CST 9359); Jak1 (CST 3344); pJak1-Tyr1034/1035 (CST 74129); Jak2 (CST 3230T); Jak3 (CST 8827); pJak3 (CST 5031); Hexokinase II (CST 2867); Enolase (Abcam ab155102); pERK-p44/42 (CST 4370); cMyc (CST 5605); Pim3 (Abcam ab71321); and β-Actin (Sigma-Aldrich, A2228).

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Protein Expression Analysis in Cells

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Tissues and cells were lysed, and protein concentrations were measured using a BCA Kit (Beyotime). The proteins were separated by SDS‐PAGE, then transferred to polyvinylidene difluoride membranes, blocked, and incubated with primary antibodies. The primary antibodies and their dilutions were as follows: β‐Actin (1:2000), hexokinase‐II (HK2, 1:1000), glucose transporter type 1 (GLUT1, 1:100,000), PKM2 (1:1000), and lactate dehydrogenase A (LDHA, 1:2000) (Abcam). Proteins were detected using an ECL kit (Affinity). Images were acquired using a Bio‐Rad image analysis system (BIO‐RAD). Quantitative image processing was performed using ImageJ software (NIH).

Chen W., Li X., Du B., Cui Y., Ma Y, & Li Y. (2022). The long noncoding RNA HOXA11‐AS promotes lung adenocarcinoma proliferation and glycolysis via the microRNA‐148b‐3p/PKM2 axis. Cancer Medicine, 12(4), 4421-4433.

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