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Igfbp3

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IGFBP3 is a protein that binds to insulin-like growth factors (IGFs), regulating their availability and activity. It plays a key role in the IGF signaling pathway. This product is intended for research use only.

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Lab products found in correlation

Tris glycine gel, by Thermo Fisher Scientific (2 mentions) Azure c500, by Azure Biosystems (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Abcam (2 mentions) Chemiluminescence reagent kit, by Thermo Fisher Scientific (1 mentions) Acetyl lysine, by Abcam (1 mentions) Ab211651, by Abcam (1 mentions) Igfbp2, by R&D Systems (1 mentions) Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions) Enhanced chemiluminescence system, by Thermo Fisher Scientific (1 mentions) P 4ebp1, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Chemilluminescence reagent kit, by Thermo Fisher Scientific (1 mentions) Epac1, by Abcam (1 mentions) Hmgb1, by Abcam (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Bicinchoninic acid kit, by Beyotime (1 mentions) E cad, by Cell Signaling Technology (1 mentions) Vimentin, by Proteintech (1 mentions)

12 protocols using igfbp3

1

Western Blot Analysis of Protein Lysates

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Whole retinal lysates from mice or cell culture lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated onto a precast Tris-glycine gel (Invitrogen) and blotted on nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, and 0.1% Tween-20) and 5% (w/v) bovine serum albumin (BSA), the membranes were treated with acetyl-lysine (1:500, Cat #80178) and IGFBP-3 (1:500, Cat# 76,001; Abcam) or GAPDH and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies overnight followed by incubation with secondary antibodies (anti-rabbit-horseradish peroxidase; Promega, Madison, WI) for 0.5–1.0 h. Antigen-antibody complexes were detected with a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA), and data were acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software. A representative blot for each treatment group is shown.
Jiang Y., Liu L, & Steinle J.J. (2018). Epac1 deacetylates HMGB1 through increased IGFBP-3 and SIRT1 levels in the retinal vasculature. Molecular Vision, 24, 727-732.
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2

Quantification of IGF and IGFBP biomarkers

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ELISA kits for Insulin-like growth factor I (IGF1, #ab211651, intra-assay coefficient of variation (CV) 2.3%) and Insulin-like growth factor-binding protein 3 (IGFBP3, #ab211652, intra-assay CV 3.1%) were purchased from Abcam (Cambridge, MA), and ELISA kit for Insulin-like growth factor-binding protein 2 (IGFBP2, #DGB200, intra-assay CV 5%) was from R&D Systems (Minneapolis, MN), and ELISA kit for Anti-mullerian hormone (AMH, #AL-124, intra-assay CV 3.7%) was purchased from AnshLabs (Webster, TX). The plasma samples from each time point of selected subjects were diluted with dilution factors of 1:80, 1:100, and 1:2000 for IGF1, IGFBP2, and IGFBP3 detection, respectively; and for AMH measurement, dilution factors of 1:10 and 1:500 were used for samples from females and males, respectively. All ELISA measurements were conducted according to manufacturer’s protocol. Duplicated wells were used to average the absorbance values and the concentration of target proteins in the plasma were calculated based on the calibration curve (R2 = 0.9848, 0.9999, 0.9936 and 0.9971 for IGF1, IGFBP2, IGFBP3, and AMH, respectively.) and dilution factor after subtraction of background.
Liu C.W., Bramer L., Webb-Robertson B.J., Waugh K., Rewers M.J, & Zhang Q. (2016). Temporal Profiles of Plasma Proteome during Childhood Development. Journal of proteomics, 152, 321-328.
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3

Western Blot Analysis of Signaling Proteins

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Cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics, Switzerland). The protein content of the cell lysates was quantified using the Bradford assay [25 (link)]. The proteins (30 μg) were resolved using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (GE Healthcare, USA). The membrane was blocked for 1 h using 5% (w/v) non-fat dry milk in Tris-buffered saline containing Tween-20 and incubated with primary and secondary antibodies according to the manufacturer’s protocol. Antibodies against p-Akt (Ser273), Akt, p-PI3K (Tyr458/Tyr199), PI3K, p-AMPK (Thr172), AMPK, p-mTOR (Ser2448), mTOR, p-p70S6K (Thr389), p70S6K, p-eukaryotic translation initiation factor 4E-binding protein 1; p-4E-BP1 (Thr37/46), 4E-BP1 (Cell Signaling Technology, USA), IGFBP3 (Abcam, UK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, USA) were used. An enhanced chemiluminescence system (Thermo Fisher Scientific, USA) was used to visualize the bands using the ChemiDoc MP system (Bio-Rad, USA). Densitometry of the bands was performed using the ImageJ software (National Institutes of Health,USA). Protein levels were quantitatively analyzed and normalized to GAPDH.
Jang H.J., Lee S., Hong E., Yim K.J., Choi Y.S., Jung J.Y, & Kim Z.H. (2022). Mychonastes sp. 246 Suppresses Human Pancreatic Cancer Cell Growth via IGFBP3-PI3K-mTOR Signaling. Journal of Microbiology and Biotechnology, 33(4), 449-462.
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4

Quantitative Immunoblotting of Retinal Proteins

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Retinal cell lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated onto a pre-cast tris-glycine gel (Invitrogen, Carlsbad, CA), blotted onto nitrocellulose membrane and blocked with TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA. Membranes were treated with HMGB1, IGFBP-3, Epac1(Abcam, Cambridge, MA) or beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with horseradish peroxidase labeled secondary antibodies. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) and data was acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software.
Liu L., Patel P, & Steinle J.J. (2018). PKA regulates HMGB1 through activation of IGFBP-3 and SIRT1 in human retinal endothelial cells cultured in high glucose. Inflammation research : official journal of the European Histamine Research Society ... [et al.], 67(11-12), 1013-1019.
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5

Western Blot Profiling of EMT Markers

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The protocol used for western blotting is described elsewhere.22 (link) Total cellular protein was extracted in lysis buffer by radioimmunoprecipitation assay. The protein concentration was determined by using the bicinchoninic acid kit (Biyuntian, Jiangsu, China). Load the same amount of protein onto 10% SDS-polyacrylamide gel electrophoresis gel. After electrophoresis, the protein was blotted onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% skim milk and incubated with the primary antibody overnight at 4 °C, and then treated with secondary antibody at 37 °C for 2 h. The primary rabbit anti-human antibodies used were as follows: ECAD (Cell Signaling Technology), NCAD (Cell Signaling Technology), vimentin (Proteintech Group), MMP1 (Cell Signaling Technology), MMP3 (Abcam), IGFBP3 (Abcam), β-catenin (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho-Akt (Cell Signaling Technology), GSK-3β (Cell Signaling Technology), phospho-GSK-3β (Cell Signaling Technology), TBP (Proteintech Group) and β-actin (Cell Signaling Technology).
Song M., Pan Q., Yang J., He J., Zeng J., Cheng S., Huang Y., Zhou Z.Q., Zhu Q., Yang C., Han Y., Tang Y., Chen H., Weng D.S, & Xia J.C. (2020). Galectin-3 favours tumour metastasis via the activation of β-catenin signalling in hepatocellular carcinoma. British Journal of Cancer, 123(10), 1521-1534.
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6

Quantification of Angiogenic Factors

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To confirm the semi-quantitative results of the angiogenesis array, protein concentrations of selected pro- and anti-angiogenic factors were measured in the CM, EV-depleted CM and (lysed) EVs of DPSCs and BM-MSCs by ELISA. ELISA kits for VEGF (#DY293B), insulin-like growth factor-binding protein-1 (IGFBP-1, #DY871), TIMP-1 (#DY970), angiopoietin-1 (Angpt-1, #DY923) (R&D Systems) and IGFBP-3 (#ab217607, Abcam) were used conform the manufacturer’s guidelines. Data were normalized to the number of seeded cells.
Merckx G., Hosseinkhani B., Kuypers S., Deville S., Irobi J., Nelissen I., Michiels L., Lambrichts I, & Bronckaers A. (2020). Angiogenic Effects of Human Dental Pulp and Bone Marrow-Derived Mesenchymal Stromal Cells and their Extracellular Vesicles. Cells, 9(2), 312.
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7

XTTF Decoction for Biomarker Analysis

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The components of XTTF decoction were purchased from Shanghai Ley’s Pharmaceutical Co., Ltd. (Shanghai, China), and the decoction was manufactured in Changzheng Hospital. Antibodies to IGF-1, IGF-1R, and IGFBP-3 were obtained from Abcam (Cambridge, UK). Trizol reagent and transcription reagent were obtained from JRDUN Biotechnology Company (Shanghai, China).
Zhao Y., Xiu L., Li Y., Lu Y., Wang X, & Wei P. (2019). Effects of Xiaotan Tongfu decoction on hepatic metastasis in obesity-associated colorectal cancer. The Journal of International Medical Research, 47(2), 915-925.
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8

Protein and Gene Expression Analysis

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Western blot and qRT-PCR were performed as described in our previous study [16 (link)]. Primary antibodies for Western blot were: PHF5A (1:500; Invitrogen); IGFBP3 (1:500; Abcam); PIK3CB (1:500; CST); AKT2 (1:500; Abcam); DDIT3 (1:200; Abcam); Skp2 (1:500; Abcam); P53 (1:1000; CST); GAPDH (1:2000; Santa Cruz Biotechnology). The primers used for qRT-PCR are listed in Additional file 1: Table S1. Gene and protein expression levels were normalized to those of the internal control GAPDH.
Yang Y., Zhu J., Zhang T., Liu J., Li Y., Zhu Y., Xu L., Wang R., Su F., Ou Y, & Wu Q. (2018). PHD-finger domain protein 5A functions as a novel oncoprotein in lung adenocarcinoma. Journal of Experimental & Clinical Cancer Research : CR, 37, 65.
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9

Western Blot Analysis of Tissue Proteins

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Tissue proteins were extracted by RIPA lysis buffer (Cell Signaling Technology, USA) containing a protease inhibitor cocktail (KeyGEN BioTECH, China). The protein concentration was determined by the BCA protein assay (Beyotime, China). After that, the proteins were isolated by SDS–PAGE and then passed on to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% bovine serum albumin at room temperature for 1 h and then incubated with primary antibodies against collagen I (Abcam, USA), matrix metalloproteinase 9 (MMP9) (Abcam, USA), IGFBP3 (Abcam, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Bcl-2-associated X protein (Bax) (Abcam, USA), and GAPDH (Abcam, USA). The membrane was washed and soaked with appropriate secondary antibodies at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Millipore, USA) and imaged with an imaging system (Tanon, China). The GAPDH signal was used as a loading control.
Hu D., Ge Y., Cui Y., Li K., Chen J., Zhang C., Liu Q., He L., Chen W., Chen J., Hu C, & Xiao H. (2022). Upregulated IGFBP3 with Aging Is Involved in Modulating Apoptosis, Oxidative Stress, and Fibrosis: A Target of Age-Related Erectile Dysfunction. Oxidative Medicine and Cellular Longevity, 2022, 6831779.
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10

Graphene Oxide and Antibody Assay

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GO 1% dispersion was purchased from GO Graphene William Blythe Limited (Essex, England, United Kingdom). Actin, IGF-1, IGFBP3, and Nrf-2 antibodies were purchased from Abcam (Cambridge, MA, USA).
Burnett M., Abuetabh Y., Wronski A., Shen F., Persad S., Leng R., Eisenstat D, & Sergi C. (2020). Graphene Oxide Nanoparticles Induce Apoptosis in wild-type and CRISPR/Cas9-IGF/IGFBP3 knocked-out Osteosarcoma Cells. Journal of Cancer, 11(17), 5007-5023.
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