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Igfbp 2

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IGFBP-2 is a protein that binds to insulin-like growth factors (IGFs) and regulates their bioavailability and activity. It is a key component of the IGF signaling pathway.

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Lab products found in correlation

P akt, by Cell Signaling Technology (2 mentions) E cadherin, by Cell Signaling Technology (2 mentions) Igf1r, by Santa Cruz Biotechnology (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Cd163, by Santa Cruz Biotechnology (1 mentions) Foxa1, by Abcam (1 mentions) P mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions) Clarity ecl substrate, by Bio-Rad (1 mentions) Image lab software, by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Fibronectin, by BD (1 mentions) Gapdh, by Merck Group (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Vimentin, by BD (1 mentions) Ptch1, by Santa Cruz Biotechnology (1 mentions) N cadherin, by Cell Signaling Technology (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Igfbp2, by Abcam (1 mentions) Mouse elisa kit, by Abcam (1 mentions)

8 protocols using igfbp 2

1

Quantification of IGFBP Proteins in Hepatocytes

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IGFBP2 protein abundance was examined in hepatocyte cell lysates by Western blotting [21 (link)] using IGFBP2 (Santa Cruz Biotechnology, Heidelberg, Germany) and GAPDH (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. IGFBP2, IGFBP3, and IGF1 content was measured in unprocessed culture supernatants obtained from 24 h serum-free hepatocyte cultures, as well as in blood plasma using specific mouse Elisa kits (Abcam, Cambridge, UK/R&D systems, Wiesbaden, Germany).
Fahlbusch P., Knebel B., Hörbelt T., Barbosa D.M., Nikolic A., Jacob S., Al-Hasani H., Van de Velde F., Van Nieuwenhove Y., Müller-Wieland D., Lapauw B., Ouwens D.M, & Kotzka J. (2020). Physiological Disturbance in Fatty Liver Energy Metabolism Converges on IGFBP2 Abundance and Regulation in Mice and Men. International Journal of Molecular Sciences, 21(11), 4144.
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2

IHC and Flow Cytometry Analysis of PDAC Tumor Samples

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The use of patient-derived material was approved by the institutional research ethics committee at Tianjin Medical University General Hospital (TMUGH). Tumor samples were obtained from 96 patients who underwent surgical resection at the hospital between 2009 and 2018 and had a histological diagnosis of PDAC. Following informed consent, the patient’s demographic and clinical characteristics were recorded and analyzed. Consecutive sections of formalin-fixed, paraffin embedded tumor samples were subjected to IHC for IGFBP2, CD163, FOXP3, Ki-67, and phospho-(Y705)-STAT3 (Santa Cruz Biotechnology). The results were scored by two pathologists who were blinded to the clinicopathologic data. Fresh PDAC patient surgical samples from TMUGH were processed into single-cell suspensions with 1 mg/ml collagenase, 2.5 U/ml hyaluronidase and 0.1 mg/ml DNase and were subjected to flow cytometry for cell surface markers with antibodies to human CD4, CD25, FOXP3, CD8, CD45, CD68, and CD163 (BD Biosciences). For each tumor, the mirror image tumor sample was also collected and processed to FFPE samples for measuring the IGFBP2 expression by IHC.
Sun L., Zhang X., Song Q., Liu L., Forbes E., Tian W., Zhang Z., Kang Y., Wang H., Fleming J.B., Pasche B.C, & Zhang W. (2020). IGFBP2 promotes tumor progression by inducing alternative polarization of macrophages in pancreatic ductal adenocarcinoma through the STAT3 pathway. Cancer letters, 500, 132-146.
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3

Comprehensive Western Blot Analysis

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Western blot analysis was performed as described previously [40 (link)]. Briefly, 30 μg of protein were run on 10% SDS-PAGE, transferred to nitrocellulose membrane (BioRad, Hertfordshire, UK) and immunoblotted with the following antibodies: E-cadherin (1:1000, Cell Signaling Hertfordshire, UK), vimentin (1:500, BD Biosciences Oxford, UK), fibronectin (1:500, BD Biosciences Oxford, UK), FOXA1(1:100, Abcam), IGFBP-2(1:1000, Santa Cruz Heidelberg, Germany), MAPK (Erk1/2)(1:1000, Cell Signaling Hertfordshire, UK), AKT (1:1000, Cell Signaling Hertfordshire, UK), p-AKT (S473)(1:1000, Cell Signaling Hertfordshire, UK), p-IGF1-R/IR (1:1000, Cell Signalling), p- MAPK (Erk1/2; Thr202/Tyr204)(1:1000, Cell Signaling Hertfordshire, UK), GAPDH (1:5000, Merck Millipore Hertfordshire, UK) and tubulin (1:5000, Merck Millipore Hertfordshire, UK), following the manufacturer’s instructions. After incubation with specific secondary antibodies conjugated to peroxidase (Sigma-Aldrich, Dorset, UK.), proteins were visualised by Clarity ECL substrate (BioRad, Hertfordshire, UK) using BioRad Chemidoc XRS + system and analysed using Image lab software (BioRad, Hertfordshire, UK).
Mansor R., Holly J., Barker R., Biernacka K., Zielinska H., Koupparis A., Rowe E., Oxley J., Sewell A., Martin R.M., Lane A., Hackshaw-McGeagh L, & Perks C. (2020). IGF-1 and hyperglycaemia-induced FOXA1 and IGFBP-2 affect epithelial to mesenchymal transition in prostate epithelial cells. Oncotarget, 11(26), 2543-2559.
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4

Gemcitabine and Signaling Pathway Evaluation

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Gemcitabine was purchased from Eli Lily (France). The following antibodies were used in this study: IGFBP-2, PTCH-1and Gli1 (Santa Cruz Biotechnology, CA, USA); E-cadherin, N-cadherin and vimentin (Cell Signaling Technology, Inc., MA, USA); and Ki-67 (Abcam Inc., MA, USA).
Liu H., Li L., Chen H., Kong R., Pan S., Hu J., Wang Y., Li Y, & Sun B. (2017). Silencing IGFBP-2 decreases pancreatic cancer metastasis and enhances chemotherapeutic sensitivity. Oncotarget, 8(37), 61674-61686.
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5

Investigating IGF-1 Signaling Pathway

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Wy14643 (Sigma–Aldrich) and recombinant human IGF-1 (Life Technologies) were dissolved in the recommended solvents. Antibodies against p-IGF-1R, p-Akt, Akt, p-mTOR and p-S6 kinase (S6K) were purchased from Cell Signaling Technology and anti-PPARα, IGFBP-2, IGF-1R and β-actin were from Santa Cruz Biotechnology.
Kang H.S., Kim M.Y., Kim S.J., Lee J.H., Kim Y.D., Seo Y.K., Bae J.H., Oh G.T., Song D.K., Ahn Y.H, & Im S.S. (2015). Regulation of IGFBP-2 expression during fasting. Biochemical Journal, 467(Pt 3), 453-460.
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6

Western Blot Analysis of Signaling Pathways

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Cells were rinsed once using cold 1×PBS buffer, lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma) and centrifuged at 14,000 rpm, 4°C for 15 minutes. Total cellular protein was analyzed by SDS-PAGE using 4–12% NuPAGE Gel (Invitrogen), transferred to nitrocellulose membrane and blocked with 5% Bovine Serum Albumin (Sigma) for 1h at room temperature. Immunoblotting was performed by using primary antibody and respective secondary antibodies, and detected using Supersignal West Pico Chemiluminescent substrate (Thermo Scientific). Antibodies were procured from: Phospho-MAPK (T202/Y204), Phospho-S6 (S235/236) (Cell Signaling Technology); IGFBP2, ERα, tuberin (Santa Cruz), and beta-actin (Sigma).
Li X., Liu X., Zhang L., Li C., Zhang E., Ma W., Fan Q, & Yu J.J. (2017). Insulin growth factor binding protein 2 mediates the progression of lymphangioleiomyomatosis. Oncotarget, 8(22), 36628-36638.
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7

Protein Expression Analysis of Liver Tissues

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Proteins were isolated from liver tissues and analyzed according to the methods described previously. The membranes were probed with antibody against phospho-AMPK, AMPK, phospho-IGF-1R, phospho-Akt, Akt, phospho-Sirt1, Sirt1, phospho-p70S6K, p70S6K (Cell Signaling Technology, Danvers, MA, USA), PPARα, IGFBP-2, IGF-1R, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and then developed using an ECL Western blot detection kit (Amersham Bioscience, Piscataway, NJ, USA).
Kang H.S., Cho H.C., Lee J.H., Oh G.T., Koo S.H., Park B.H., Lee I.K., Choi H.S., Song D.K, & Im S.S. (2016). Metformin stimulates IGFBP-2 gene expression through PPARalpha in diabetic states. Scientific Reports, 6, 23665.
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8

Protein Expression Analysis in Cell Lysates

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Cell lysates and media were run on 12% SDS-PAGE gel and proteins transferred to a Hybond-C nitrocellulose membrane (GE Healthcare, Bucks, UK). Proteins were probed with anti-insulin-like growth factor binding protein-2 (IGFBP-2) 1:1000 (sc-6001 Santa Cruz); anti-ERα 1:750 (sc-73479 Santa Cruz, TX, USA); anti-PARP 1:1000 (556494 BD, Oxford, UK); anti-GAPDH 1:5000 (MAB 374 Millipore, Darmstadt, Germany); anti-α-tubulin 1:5000 (ABJ1178 Autogen Bioclear, Wiltshire, UK); anti-Her2 1:1000 (#2248 Cell Signaling, Hertfordshire, UK); anti-IGF-I receptor (IGF-IR) 1:1000 (D23H3 Cell Signaling, Hertfordshire, UK); anti-p53 1:1000 (sc-126 Santa Cruz, TX, USA); anti-p21 1:2000 (05-345 Upstate Biotechnology, New York, NY, USA); or anti-β-actin 1:10000 (A5441 Sigma-Aldrich, Gillingham, Dorset, UK) following the manufacturer’s instructions. Secondary antibodies were diluted in 5% milk-TBST (20 mM Tris, 136 mM sodium chloride, 0.1% Tween-20, pH 7.4) and proteins visualized using supersignal west dura ECL solution (Thermo Fischer, Ulm, Germany) and the UVP Chemi-Doc-IT imaging system (Bio-Rad, Hertfordshire, UK), as described previously (20 (link)).
Zeng L., Holly J.M, & Perks C.M. (2014). Effects of Physiological Levels of the Green Tea Extract Epigallocatechin-3-Gallate on Breast Cancer Cells. Frontiers in Endocrinology, 5, 61.
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