Total proteins were extracted from homogenized tissues and cell lysates using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, Rockford, USA). An equal amount of protein lysates was separated on 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After an overnight incubation with the following primary antibodies at 4 °C: HOXB5 (1:1000, #109375, Abcam, Cambridge, UK), SESN3 (1:800, #97792, Abcam), IGF1R (0.1 μg/mL, #AF-305-SP, R&D Systems, Inc., Minneapolis, USA), protein kinase B (Akt, 1:1000, #9272, Cell Signaling), phosphorylated Akt (p-Akt, 1:1000, #9271, Cell Signaling, Danvers, USA), PCNA (PCNA, 1:1000, #18197, Abcam), p27 (1:1000, #32034, Abcam), B-cell lymphoma 2 (Bcl-2, 1:800, #59348, Abcam), E-cadherin (1:2000, #40772, Abcam), N-cadherin (1:1000, #76057, Abcam), cleaved caspase-3 (1:1000, #49822, Abcam), and GAPDH (1:2000, #37168, Abcam), the membranes were washed with Tris-Buffered Saline and stained with secondary antibody (1:2000, #6721, Abcam) for 60 min at room temperature. The images were captured using Alphalmager™ 2000 Imaging System (Alpha Innotech, USA).
Igf1r
Manufactured by R&D Systems
Sourced in United Kingdom, United States
IGF1R is a laboratory equipment product that functions as an insulin-like growth factor 1 receptor. It is a protein that plays a role in cellular signaling processes.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Abcam (1 mentions)
Hoxb5, by Abcam (1 mentions)
Protein kinase b akt, by Cell Signaling Technology (1 mentions)
Phosphorylated akt p akt, by Cell Signaling Technology (1 mentions)
Alphalmager 2000 imaging system, by Bio-Techne (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Elisa, by R&D Systems (1 mentions)
Collagenase d, by Roche (1 mentions)
Ez link nhs biotin, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Western lightning chemiluminescent kit, by PerkinElmer (1 mentions)
4 protocols using igf1r
1
Western Blot Analysis of Cellular Proteins
2
Quantifying Hyaluronic Acid Production in Fibroblast-Macrophage Cultures
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Hyaluronic acid (HA) production was quantified by ELISA following manufacturer’s guidance (R&D Systems, MN, USA). Fibroblasts, with or without macrophages, were cultured in collagen gels in serum-free medium with or without 5 μg/ml IGF-1R blocking antibody 1H7. After 3 days, the culture medium was collected and the gels digested with 0.05% collagenase-D (Roche, UK) for 30 min at 37 °C to, extract any further HA trapped in the gel. The collagenase digestion was centrifuged to remove cells and debris and the supernatant collected. The mixture of media and supernatant from digested gels was diluted in serum-free DMEM (1:30), and assayed according to the manufacturer’s instructions. Gels without cells, and gels with macrophages only, were used for baseline calibration. To confirm the efficiency of the blocking antibody, HA levels were measured in fibroblasts cultured in serum-free medium with 20 μM IGF-1 (R&D Systems, MN, USA) to stimulate HA production, with or without IGF-1R blocking antibody.
3
Biolayer Interferometry for Bispecific Binding
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Biolayer light interferometry (BLI) was performed using an Octet RED96 instrument (ForteBio; Pall Life Sciences). Bispecific binding was confirmed by first capturing biotin labeled HER2 or EGFR (R&D System) (ligand 1) at 10 µg/mL onto Streptavidin biosensors for 180 s. The biosensors were then submerged in binding buffer (PBS/0.2% polyethylene glycol sorbitan monolaurate (TWEEN 20) for a wash for 60 s followed by immersion in a solution containing 500 nM of either the parental or bsAbs for 120 s, followed by another wash and subsequent immersion in a solution containing 30 μg/mL of either EGFR or IGF1R (R&D System) (ligand 2) for 120 s for each respective dual binding assay. Biotinylation of probes was performed using EZ-Link NHS-Biotin (ThermoFisher) following the manufacturer’s specifications.
4
Western Blot Analysis of IGF Axis Proteins
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Equal amounts of protein lysates from MSCs were resolved by either 6% or 15% SDS/PAGE and electroblotted onto polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Nonspecific binding sites were blocked for 1 h with PBS containing 5% BSA and 0.1% Tween‐20 at room temperature (RT). The membrane was then immunoblotted against anti‐IGFBP2 clone C‐18 (dilution 1 : 1000; Santa Cruz Biotechnology Inc.), IGF‐2 (dilution 1 : 500; R&D Systems), IGF‐1R and IGF‐2R (dilution 1 : 200; R&D Systems) at 4 °C. Following washing and incubation with either rabbit anti‐goat or goat anti‐mouse horseradish peroxidase‐conjugated secondary antibodies (dilution of either 1 : 5000 or 1 : 10 000; DakoCytomation, Denmark), proteins of interest were visualized with an enhanced chemiluminescence using Western Lightning chemiluminescent kit (Perkin‐Elmer, MA, USA). Normalization was performed using antibody against actin (dilution 1 : 20 000; Thermo Fisher Scientific, Fremont, CA) or tubulin (dilution 1 : 5000; Santa Cruz Biotechnology Inc.). Semiquantitation analysis, to normalize the protein bands against respective loading controls, was performed using Metavue software v6.1 (Molecular Devices, LLC, CA).
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