Recombinant human igf2

Manufactured by R&D Systems

Sourced in United States

Recombinant human IGF2 is a polypeptide growth factor produced in a mammalian cell expression system. IGF2 is involved in the regulation of growth and development.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (2 mentions) Epidermal growth factor (egf), by Merck Group (1 mentions) Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions) Il 20, by R&D Systems (1 mentions) Heparin, by STEMCELL (1 mentions) Ptch1, by Cell Signaling Technology (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Bcl 2, by Cell Signaling Technology (1 mentions) Prism 8, by GraphPad (1 mentions) Fibronectin, by Santa Cruz Biotechnology (1 mentions) α sma antibody, by Merck Group (1 mentions) Gapdh antibody, by Santa Cruz Biotechnology (1 mentions) Scrambled sirna, by Thermo Fisher Scientific (1 mentions) Igf2r, by Cell Signaling Technology (1 mentions) Igf1r, by Thermo Fisher Scientific (1 mentions) Anti rabbit igg hrp, by GE Healthcare (1 mentions) Hrp conjugated anti mouse igg, by Promega (1 mentions) Phospho smad, by Cell Signaling Technology (1 mentions) Total smad, by Cell Signaling Technology (1 mentions)

8 protocols using recombinant human igf2

Quantifying Recombinant Human IGF2 Using ELISA

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Recombinant human IGF2 (from R and D Systems) was coated on ELISA plates at 50 ng/well in 50 ul PBS overnight. m67 standard curve was established by adding known amount of m67 to cynomolgus plasma collected from an unrelated animal, from 5 nM to 0.039 nM. The test plasma were diluted into 1:100 (for early samples) or 1:3 (late samples) to make sure the final reading falls in the linear range of the m67 standard curve. After the wells were blocked with 2% non-fat dry milk in PBS (MPBS), m67 serially diluted solutions and test plasma were added to wells in duplicates. The ELISA plates were incubated at 37°C for 2 hours, and washed with PBS+0.05% Tween 20 (PBST) for 4 times. To detect bound m67 on plate, a goat anti-human Fc antibody coupled with horse radish peroxidase (Jackson ImmunoResearch Lab) was used at 1:1000. After wash, the substrate ABTS was added and the color reaction was monitored at 405 nm wavelength. The concentration of tested plasma was calculated from the standard curve of m67. The averages from 3 monkeys were plotted on graph.

Feng Y., Zhao Q., Chen W., Wang Y., Crowder K, & Dimitrov D.S. (2014). A New Bispecific Antibody Targeting Non-overlapping Epitopes on IGF2: Design, in vitro Characterization and Pharmacokinetics in Macaques. Experimental and molecular pathology, 97(3), 359-367.

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Sphere Formation Assay for Patient-Derived Tumor Cells

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Patient-derived tumor tissues were treated according to a previously described method.^{16 (link)} We previously confirmed that patient-derived tumor cells plated at 5000 cells per ml yield tumor spheres that are clonally derived from single cells.^{16 (link)} Sphere formation assay was performed as described previously.^{16 (link)} Briefly, cells were plated as single cells on ultralow attachment 24-well plates (2000–5000 cells per well). Spheres were grown in SCM containing 20 ng/ml EGF (Millipore, Billerica, MA, USA), 20 ng/ml basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA), B27 (Gibco, Grand Island, NY, USA) and heparin (Stem Cell Technologies, Vancouver, BC, Canada) or in DMEM/F-12 medium supplemented with 20 ng/ml recombinant human HRG (R&D Systems Inc., Lille, France), 200 ng/ml recombinant human IGF2 (R&D Systems Inc.) and 20 ng/ml AR (R&D Systems Inc.) or 200 ng/ml IL-20 (R&D Systems Inc.), with or without anti-IGF2 antibody (clone S1F2; Millipore) and anti-AR antibody (R&D Systems Inc.). Spheres >75 μm in diameter were counted after 4–7 days. Sphere-forming efficiency was calculated as the ratio of the number of spheres formed to the number of cells originally plated (2000–5000 cells per well).

Tominaga K., Shimamura T., Kimura N., Murayama T., Matsubara D., Kanauchi H., Niida A., Shimizu S., Nishioka K., Tsuji E.I., Yano M., Sugano S., Shimono Y., Ishii H., Saya H., Mori M., Akashi K., Tada K.I., Ogawa T., Tojo A., Miyano S, & Gotoh N. (2016). Addiction to the IGF2-ID1-IGF2 circuit for maintenance of the breast cancer stem-like cells. Oncogene, 36(9), 1276-1286.

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Antibody-based Signaling Pathway Analysis

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Antibodies against CD44 and IGF2BP3 were purchased from Santa Cruz (Dallas, TX, USA). Antibodies of Bax, bcl‐2, caspase‐3, Gli2, Hhip, Ptch1 and Ptch were obtained from Cell Signaling Technology. All other chemicals were purchased from Sigma‐Aldrich (Saint Louis, MO, USA). Recombinant human IGF2 was purchased from R&D (Minneapolis, MN, USA).

Liu Y., Yu C., Wu Y., Sun X., Su Q., You C, & Xin H. (2017). CD44+ fibroblasts increases breast cancer cell survival and drug resistance via IGF2BP3‐CD44‐IGF2 signalling. Journal of Cellular and Molecular Medicine, 21(9), 1979-1988.

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Cell Viability Assay for Anticancer Drugs

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Cells were seeded as 5×10⁴ cells/well in 96-well plates and, after 24 hours, exposed to 10 increasing concentrations of cisplatin, doxorubicin, CM-272, decitabine (5-azacytidine) (all from Selleckchem) and BIX-01294 trihydrochlorid hydrate (Sigma-Aldrich) ranging from 5 nM to 100 µM with DMSO. Rescue experiments on CM-272 using 10 ng/mL recombinant human IGF2 (RD Systems, Minneapolis, MN) were done for 48 hours by refreshing media after 24 hours. Following 48 hours of incubation, cell viability was measured using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays and absorbance measurement as described.²¹ (link) Half-maximal inhibitory concentrations (IC50) and AUC values were calculated by plotting nonlinear regression of drug response curves using GraphPad Prism 8 (GraphPad Software, San Diego, CA).

Demir S., Razizadeh N., Indersie E., Branchereau S., Cairo S, & Kappler R. (2024). Targeting G9a/DNMT1 methyltransferase activity impedes IGF2-mediated survival in hepatoblastoma. Hepatology Communications, 8(2), e0378.

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IGF-II Signaling Pathway Regulation

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DMEM for cell culture was purchased from Corning (Corning, NY, USA). Fetal bovine serum, protease inhibitor cocktail, and αSMA antibody were from Sigma-Aldrich (St. Louis, MO, USA). IGF1R inhibitor I-OMe-Tyrphostin AG 538 was from Calbiochem (San Diego, CA, USA). Recombinant human IGF-II and anti-IGF-II antibody were purchased from R&D Systems (Minneapolis, MN, USA). Collagen, Fibronectin, and GAPDH antibodies were from Santa Cruz (Santa Cruz, CA, USA). IGF1R, IGF2R, IR-β, phospho-SMAD and total SMAD antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine^® 2000 was purchased from Invitrogen (Carlsbad, CA, USA). IGF1R, IR, and isotype control antibodies used in neutralization experiments were obtained from GroPep Bioreagents (Thebarton SA 5031, Australia). IGF1R, IGF1R, IR, and scrambled siRNA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HRP-conjugated anti-mouse IgG was from Promega (Madison, WI, USA) and anti-rabbit-HRP IgG was from GE Healthcare Life Sciences (Chicago, IL, USA).

Garrett S.M., Hsu E., Thomas J.M., Pilewski J.M, & Feghali-Bostwick C. (2019). Insulin-like growth factor (IGF)-II- mediated fibrosis in pathogenic lung conditions. PLoS ONE, 14(11), e0225422.

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Inhibition of Enzyme Uptake in Brain Cells

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Uptake inhibition assays were performed in MPS IIIB fibroblasts and two brain tumor-derived cell lines, Dao Y and Es (cerebellar medulloblastoma and glioblastoma, respectively, kindly provided by Dr. J Lasky, Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, CA). Cells were seeded in 12-well plates and grown to confluence. Culture medium was replaced with 0.5 ml of EMEM without serum prior to the addition of uptake inhibitors. 5 µg/ml recombinant human IGF-II (R&D Systems; Minneapolis, MN) or 5 mM D-mannose 6-phosphate (Sigma-Aldrich; St. Louis, MO) was applied to the cells for 10 minutes prior to applying purified rhNAGLU-IGF-II at a final concentration of 160 units/ml. Following 4 hours incubation at 37°C and 5% CO₂, cells were harvested and intracellular NAGLU activity was measured as described above. The enzyme activity measured in MPS IIIB fibroblasts with rhNAGLU-IGF-II treatment alone was defined as 100% and the intracellular enzyme activity observed in the presence of inhibitor(s) was normalized to rhNAGLU-IGF-II activity. The endogenous intracellular enzyme activity measured in brain tumor-derived cell lines was defined as 100% and the enzyme uptake with or without inhibitor was normalized to this value.

Kan S.H., Troitskaya L.A., Sinow C.S., Haitz K., Todd A.K., Di Stefano A., Le S.Q., Dickson P.I, & Tippin B.L. (2014). Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts. The Biochemical journal, 458(2), 281-289.

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Cell Signaling Pathway Analysis

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Kaempferol, essentially fatty acid-free bovine serum albumin, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 7-amino-actinomycin D (7-AAD), phosphatidylinositol, transferrin and anti-β-actin antibody were obtained from Sigma (St. Louis, MO, USA); selenium and DMEM/Ham’s F-12 nutrient mixture (DMEM/F-12 were purchased from Gibco BRL (Gaithersburg, MD, USA)); [methyl-³H]thymidine, protein A-Sepharose and horse-radish peroxidase-conjugated anti-rabbit and anti-mouse IgG were from Amersham (Arlington Heights, IL, USA); antibodies against HRG, IGF-IRβ, p85 and ErbB3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Akt, phospho (P)-Akt, ERK-1/2 and P-ERK-1/2 were from Cell Signaling Technology (Beverly, MA, USA); anti-phosphotyrosine-RC20 (PY20) antibody linked to horse-radish peroxidase was purchased from BD Transduction Laboratories (Palo Alto, CA, USA); anti-IGF-II antibody was from Amano International Enzyme (Troy, VA, USA); recombinant human HRG-β and recombinant human IGF-II were purchased from R&D Systems, (Minneapolis, MN, USA); phycoerythrin-conjugated Annexin V was from BD Pharmingen, (Franklin Lake, NJ, USA); and [γ-³²P]ATP was obtained from PerkinElmer Life and Analytical Sciences, Inc. (Boston, MA, USA).

Lee H.S., Cho H.J., Kwon G.T, & Park J.H. (2014). Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells. Journal of Cancer Prevention, 19(3), 161-169.

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Quantification of IGFBP-4 and PAPP-A

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Cell culture components were from Gibco Life Technologies. Bovine serum albumin (BSA) was from MilliporeSigma. Recombinant human IGF-II was from R&D Systems and IGFBP-4 was a gift from our colleague, Professor Claus Oxvig (Aarhus University, Denmark). Anti-IGFBP-4 antibodies were purchased from Abcam: N-terminal ab92625 and C-terminal ab77350. Secondary antibodies were from LiCor, Inc: Donkey anti-goat (926–68074) and donkey anti-rabbit (926–32213). Etoposide was obtained from Enzo Life Sciences. picoPAPP-A ELISA kits and monoclonal antibody generated against a unique exosite in PAPP-A (mAb-PA1/41)^{38 (link), 39 (link)} were obtained from Ansh Labs (Webster, TX).

Conover C.A, & Bale L.K. (2022). Senescence Induces Proteolytically-active PAPP-A Secretion and Association with Extracellular Vesicles in Human Pre-adipocytes. Experimental gerontology, 172, 112070.

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