Human igf1

Manufactured by Merck Group

Sourced in United States

Human IGF1 is a laboratory research product produced by Merck Group. It is a recombinant human insulin-like growth factor 1 protein. The core function of Human IGF1 is to facilitate research and development related to the insulin-like growth factor 1 signaling pathway.

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8 protocols using human igf1

Hippocampal Slice Culture Maintenance

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Unless stated otherwise, cell culture media and supplements were purchased from Invitrogen (Darmstadt, Germany), human IGF1 from Sigma-Aldrich (Seelze, Germany) and Millicell cell culture inserts (0.4 μm, 30 mm diameter) for hippocampal slice cultures from Merck Millipore (Darmstadt, Germany). PTX and LY294007 were purchased from Enzo Life Sciences (Lörrach, Germany). All other biochemicals and chemicals were provided in analytical grade purity from Roth (Karlsruhe, Germany) or Sigma-Aldrich. Plasticware was purchased from Corning Life Sciences (Wiesbaden, Germany) or Greiner Bio-One (Frickenhausen, Germany).

Milosch N., Tanriöver G., Kundu A., Rami A., François J.C., Baumkötter F., Weyer S.W., Samanta A., Jäschke A., Brod F., Buchholz C.J., Kins S., Behl C., Müller U.C, & Kögel D. (2014). Holo-APP and G-protein-mediated signaling are required for sAPPα-induced activation of the Akt survival pathway. Cell Death & Disease, 5(8), e1391-.

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Investigating IGF Signaling Regulation

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DMSO, doxycycline, cycloheximide, rapamycin, insulin, human IGF1, human IGF2, RPL26 and actin antibodies were from Sigma-Aldrich (St. Louis, Missouri, USA); Torin 1 from TOCRIS. Antibodies: P-AKT(Ser473), AKT, pS6K(Thr389), S6K, P-4E-BP1(Thr37/460), 4E-BP1, P-AMPK, AMPK, IGF1R, IRS1, IRS2, P21, Phos-Tyrosine from Cell Signaling Technology; Grb14 and IGFBP2 antibodies from abcam. IMP antibodies were described previously (Dai et al., 2011 (link); Dai et al., 2013 (link)). The inducible Lentiviral shRNA for Grb14, IGFBP2, p21 and HMGA1 were from Dharmacon and the stably expressed Flag-IMP2 variants and Flag-HMGA1were generated using pcDNA6/TR and pcDNA5/TO vectors; these reagents were used according to manufacturer’s instructions. The P-site mutants of IMP2 were generated using the QuikChange site-directed mutagenesis kit (Stratagene).

Dai N., Ji F., Wright J., Minichiello L., Sadreyev R, & Avruch J. (2017). IGF2 mRNA binding protein-2 is a tumor promoter that drives cancer proliferation through its client mRNAs IGF2 and HMGA1. eLife, 6, e27155.

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IGF1 and IGF2 Acquisition Protocol

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Human IGF2 was purchased from GroPep Bioreagents (Adelaide, Australia) and human IGF1 was purchased from Sigma-Aldrich.

Potalitsyn P., Selicharová I., Sršeň K., Radosavljević J., Marek A., Nováková K., Jiráček J, & Žáková L. (2020). A radioligand binding assay for the insulin-like growth factor 2 receptor. PLoS ONE, 15(9), e0238393.

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Signaling Pathways in Epithelial and Fibroblast Cells

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NMuMG epithelial cells and wild-type and Tgfbr1^−/− (102 (link)) mouse embryonic fibroblasts (MEFs) were cultured in DMEM with 4 mM glucose and 10% fetal bovine serum (FBS) at 37°C and 5% CO2 in a humidified incubator. Cells were transfected using Lipofectamine 2000 (Invitrogen) or RNAiMAX (Invitrogen) for double-stranded siRNA, according to the manufacturer’s protocol.
Expression plasmids encoding Flag- or Myc-tagged human TβRI or TβRII have been described (103 (link)). An expression plasmid encoding Flag-tagged AS160 was from Gustav E. Lienhard (Dartmouth Medical School, USA) (30 (link)), and a plasmid encoding NH2-terminally myristolated Akt2 was from Kevan Shokat (University California San Francisco, USA).
Insulin from bovine pancreas (Sigma) was used at 100 nM, human IGF-1(Sigma) was used at 50 ng/ml, and TGF-β1 from HumanZyme was used at 0.2 ng/ml. The TβRI kinase inhibitor SB4315242 (Sigma) was used at 5 μM and added 30 min before treatment. GDC0941 (Selleck Chemicals), Akt VIII (EMD Milipore), and U0126 (Sigma) were used at 10 μM, 5 μM, or 10 μM, respectively.

Budi E.H., Muthusamy B.P, & Derynck R. (2015). The insulin response integrates increased TGF-β signaling through Akt-induced enhancement of cell surface delivery of TGF-β receptors. Science signaling, 8(396), ra96.

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Metabolic Phenotyping and Signaling

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Comprehensive Laboratory Animal Monitoring Systems (CLAMS, Columbus instruments) and DEXA measurements were performed at the Joslin Diabetes Research Center (DRC) core. Glucose tolerance tests and insulin tolerance tests were performed as previously described (Bruning et al., 1998 (link)). Insulin levels were measured using a mouse insulin ELISA kit (Crystal Chem), triglycerides were measured using a triglyceride assay kit (Abnova), and FGF21 serum levels were measured with a mouse/rat ELISA kit (R&D Systems, Cat# MF2100). In vivo insulin and IGF-1 signaling were performed in anesthetized, overnight-fasted mice by injecting either 5 U of regular insulin or 1 mg/kg of human IGF-1 (Sigma) via inferior vena cava (IVC) and 10-15 minutes later harvesting of tissues and snap freezing in liquid nitrogen. Lactate levels were measured in gastrocnemius at the Mayo Clinic Metabolomics Resource Core using TOF mass spectrometry.

O'Neill B.T., Lauritzen H.P., Hirshman M.F., Smyth G., Goodyear L.J, & Kahn C.R. (2015). Differential Role of Insulin/IGF-1 Receptor Signaling on Muscle Growth and Glucose Homeostasis. Cell reports, 11(8), 1220-1235.

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IGF-1R and MAPK Signaling Assay

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IGF-1Rß, phospho-IGF-1R (Tyr1135/1136), phosphor-p44/42 MAPK (Thr202/Tyr204) and phospho-Akt (Ser473) mAbs were purchased from Cell Signaling Technology. Anti-ß-arrestin1 and anti-ß-arrestin2 mAbs were from Abcam. Anti-GAPDH mAb was from Santa Cruz. Human IGF-1 and metformin were purchased from Sigma. Figitumumab was provided by Pfizer.

Cao H., Dong W., Shen H., Xu J., Zhu L., Liu Q, & Du J. (2015). Combinational Therapy Enhances the Effects of Anti-IGF-1R mAb Figitumumab to Target Small Cell Lung Cancer. PLoS ONE, 10(8), e0135844.

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Endocrine Hormone Preparation Protocol

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Rat prolactin and growth hormone were provided by the National Hormone and Pituitary Program (AF Parlow, National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA). Human IGF-1 and GLP-1 (Sigma-Aldrich, St. Louis, MO) were purchased commercially.

Brelje T.C., Bhagroo N.V., Stout L.E, & Sorenson R.L. (2017). Prolactin and oleic acid synergistically stimulate β-cell proliferation and growth in rat islets. Islets, 9(4), e1330234.

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Analysis of IGF-1 Receptor Signaling

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Human IGF-1 (catalog 13769) was obtained from Sigma-Aldrich. Primary antibodies used were as follows: anti-Phospho-Akt (Ser473) (catalog 4060), anti-Akt (catalog 9272), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (catalog 4377), anti-p44/42 MAPK (Erk1/2) (catalog 9102), anti-PI3 Kinase p85(catalog 4292) and anti-IGF-1 Receptor β-subunit (D23H3) (IGF-1R β, catalog 9750) from Cell Signaling Technology; anti-TRAF4 (catalog ab108991) and anti-IRS-1 (Phospho Y896, catalog ab 4873) from Abcam; anti-Ubiquitin (catalog sc-8017) and anti-GAPDH (catalog sc-32233) from Santa Cruz Biotechnology Inc; anti-IRS-1 (21HCLC) (catalog 71009) and anti-Phospho-IRS-1(Y612) (catalog 44-816G) from Thermo Fisher Scientific; anti-Phosphotyrosine (pTyr, clone 4G10, catalog 05-321) from Sigma-Aldrich. HRP-conjugated secondary anti-mouse (catalog 1706516) and anti-rabbit (catalog 1706515) antibodies were obtained from Bio-Rad. Monoclonal ANTI-FLAG M2-peroxidase (HRP) antibody (catalog 8592A) and EZview Red ANTI-FLAG M2 Affinity Gel (catalog F2426) were obtained from Sigma-Aldrich. Protein A-Agarose beads (catalog sc-2001) were obtained from Santa Cruz Biotechnology. Recombinant human IRS-1 protein (catalog ab70538) was obtained from Abcam. This truncated recombinant protein has amino acids from 600 to 1245, which includes TRAF4-targeted lysines for ubiquitination.

Yu W., Singh R., Wang Z., O’Malley B.W, & Yi P. (2021). The E3 ligase TRAF4 promotes IGF signaling by mediating atypical ubiquitination of IRS-1. The Journal of Biological Chemistry, 296, 100739.

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