Recombinant human GDF15 (R&D Systems, Minneapolis, MN, catalogue number 957-GD-025) was resuspended in water at 10ug/uL. Knockout with sgGREP1 #2 or controls in ZR-75–1 was performed as described above. 24 hours after infection with lentiviral sgRNA, cell culture media was refreshed to contain puromycin as above for antibiotic selection, and GDF15 or vehicle control was supplemented at the following concentrations: 0.01 pg/mL, 0.1 pg/mL, 1pg/mL, 10pg/mL, 100pg/mL. Thereafter cell culture media and recombinant GDF15 was refreshed every 24 hours. Cell viability was measured 7 days after lentiviral infection using the CellTiter-Glo reagent (Promega, Madison, WI).
Recombinant human gdf15
Manufactured by R&D Systems
Sourced in United States
Recombinant human GDF15 is a growth differentiation factor produced in a HEK293 cell expression system. It is a secreted protein that belongs to the transforming growth factor-beta (TGF-β) superfamily.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo reagent, by Promega (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Sb202190, by Merck Group (1 mentions)
Sb203580, by Merck Group (1 mentions)
Sb431542, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
Carbonyl cyanide m chlorophenyl hydrazone, by Merck Group (1 mentions)
Enzyme linked immunoassay, by R&D Systems (1 mentions)
Elecsys plgf, by Roche (1 mentions)
Anti β actin, by Transgene (1 mentions)
Anti stat3, by Thermo Fisher Scientific (1 mentions)
Smad2 3 antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti sox2, by R&D Systems (1 mentions)
Anti nestin, by Merck Group (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
Smad 1 5 9 antibody sampler kit, by Cell Signaling Technology (1 mentions)
Imiquimod imq, by InvivoGen (1 mentions)
Anti cd133 2, by Miltenyi Biotec (1 mentions)
Saracatinib, by Selleck Chemicals (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
6 protocols using recombinant human gdf15
1
Recombinant GDF15 Cytotoxicity Assay
2
Signaling Pathway Modulation Protocol
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SB431542, PD98059, SB203580, SB202190, oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human GDF15 containing an N-terminal 6-His tag was purchased from R&D Systems (#957-GD/CF). Lyophilized protein was reconstituted with sterile 4 mM HCl and stored at −70 °C.
3
Biomarker Quantification in Patients
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Serum concentrations of PLGF, GDF15 and HGF were determined in 834 patients. PLGF was tested using electrochemiluminescence test (ELECSYS PlGF, Roche Diagnostics, Mannheim, Germany). HGF was analysed using the enzyme-linked immunoassay obtained from R&D Systems (Minneapolis, USA), using a monoclonal antibody specific for HGF and a pre-coated microtiter plate. GDF15 was tested using a prototype electrochemiluminescence test containing a polyclonal GDF15 affinity chromatography-purified goat anti-human IgG antibody obtained from R&D Systems. For each of the measurement sets, a standard curve was generated with recombinant human GDF15 from R&D Systems.
4
Molecular Signaling Pathways in Stem Cells
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Recombinant human GDF15 (R&D Systems, Minneapolis, MN, USA), imiquimod (IMQ, InvivoGen, San Diego, CA, USA), neutralizing antibody against LIF (R&D Systems), and ERK1/2 inhibitor PD98059 (Cell Signaling Technology, Danvers, MA, USA) were used in this study. The antibodies used were as follows: anti-CD133/2 (Miltenyi Biotec, Auburn, CA, USA), anti-PROM1 (Sigma Aldrich, St. Louis, MO, USA), anti-Nestin (Merck-Millipore, Billerica, MA, USA), anti-Toll-like receptor 7 (anti-TLR7, Novus Biological Inc., Littleton, CO, USA), anti-Musashi (Merck-Millipore), anti-phospho-STAT3 (p-Tyr705, Cell Signaling Technology), anti-STAT3 (Thermo Fisher Scientific), anti-SOX2 (R&D Systems), anti-ALDH1 (Thermo Fisher Scientific), anti-β-actin (TransGen Biotech, Beijing, China), anti-GDF15 (Thermo Fisher Scientific), anti-LIF (Merck-Millipore), and anti-Fos (GeneTex, Irvine, CA, USA). The Smad2/3 Antibody Sampler Kit (Cell Signaling Technology) and Smad1/5/9 Antibody Sampler Kit (Cell Signaling Technology) were also employed.
5
Cardioprotective Effects of Factor 1 against Ischemia-Reperfusion Injury
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Example 3
To confirm the cardiomyocyte-protective effects of Factor 1 observed in the screen, neonatal rat ventricular cardiomyocytes were subjected to simulated ischemia-reperfusion injury in the presence or absence of recombinant Factor 1. Factor 1 (mouse homologue encoded by the nucleic acid sequence shown in SEQ ID NO: 7) was produced in COS7 cells as a His-tagged recombinant protein. As shown in
Ventricular cardiomyocytes were isolated from 1 to 3-day-old Sprague-Dawley rats by Percoll density gradient centrifugation. Cardiomyocytes were exposed to simulated ischemia for 180 min (glucose-free medium containing 2-deoxyglucose in 5% CO2/95% N2 atmosphere) followed by simulated reperfusion for 60 min (back to glucose-containing medium in 5% CO2/95% room air) in the absence (control) or presence of recombinant human GDF-15 (R&D Systems, known anti-apoptotic cytokine) or different concentrations of recombinant mouse Factor 1, as indicated. Cell death was assessed by in situ TdT-mediated dUTP nick end-labeling (TUNEL). N=3 independent experiments per condition; *P<0.05 vs. control.
6
Cisplatin, Doxorubicin, and Thalidomide Regulation
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Cisplatin, doxorubicin, and thalidomide were obtained from Sigma‐Aldrich (St. Louis, MO). Recombinant human GDF15 was purchased from R&D Systems (Minneapolis, MN). Polyclonal rabbit anti‐human ERK1/2 antibody, polyclonal rabbit anti‐human p38 MAPK antibody, monoclonal rabbit anti‐human JNK antibody, monoclonal rabbit anti‐human P‐JNK antibody, polyclonal goat anti‐human GDF15 antibody and polyclonal rabbit anti‐human NF‐κB antibody (Abcam, Cambridge, UK), polyclonal rabbit anti‐human P‐ERK1/2 antibody (Thermo Fisher Scientific, Waltham, MA, USA), monoclonal mouse anti‐human P‐P38MAPK antibody, monoclonal rabbit anti‐human Akt antibody, monoclonal rabbit anti‐human P‐Akt antibody, polyclonal rabbit anti‐human Src antibody, polyclonal rabbit anti‐human p‐Src antibody, and monoclonal rabbit anti‐human p‐NF‐κB antibody (Cell Signaling Technology, Beverly, MA) were used for Western blots. Monoclonal rabbit anti‐human CD31 antibody and polyclonal rabbit anti‐human paxillin antibody (Abcam, Cambridge, UK) were used for immunohistochemical analysis and immunofluorescence, respectively. Src inhibitor Saracatinib was purchased from Selleckchem (Houston, TX).
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