The same tumor lysates as RPPA were analyzed for levels of phospho-VEGFR2 and phospho-FGFR4. PathScan® phospho-VEGFR2 antibody ELISA kit and PathScan® phospho-FGFR4 ELISA kit were used, respectively (Cell Signaling Technology cat#7824 and #69193 C). A standard curve was prepared by making serial dilution of the lysate of VEGF-Treated HUVEC Lysate (BioRad cat#171Z0010) for pVEGFR2 and FGF2-treated HepG2 lysate for pFGFR4. Liver cancer cell HepG2 cells were obtained from JCRB Cell Bank and were treated with 100 ng/mL of recombinant FGF2 (R&D System cat#233-FB-025/CF) for 5 min on 37 °C. After centrifugation, cells were lysed with RPPA buffer with TurboNuclease (Accelagen cat#N0103P) and disrupted in Bioruptor Level M for 10 sec. After centrifugation, supernatants were used to make a standardization curve for pFGFR4 ELISA. The phosphoprotein levels were normalized with total protein concentrations measured by the BCA assay. The normalized concentration data of pVEGFR2 and pFGFR4 are available as Supplementary Data 5 .
Recombinant fgf2
Manufactured by R&D Systems
Sourced in United Kingdom
Recombinant FGF2 is a laboratory protein product. It is a member of the fibroblast growth factor family, which are involved in various cellular processes. The core function of Recombinant FGF2 is to stimulate cell growth and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Azd4547, by Chemietek (2 mentions)
Mission shrna, by Merck Group (2 mentions)
Effectene, by Qiagen (2 mentions)
Mg63 human osteosarcoma cells, by American Type Culture Collection (2 mentions)
Fibroblast growth factor 2 (fgf2), by Bio-Rad (1 mentions)
Heparin from porcine intestinal mucosa, by Merck Group (1 mentions)
Recombinant fgf10, by R&D Systems (1 mentions)
Heparitinase 1, by Seikagaku (1 mentions)
Nestin, by Santa Cruz Biotechnology (1 mentions)
Notch3, by Santa Cruz Biotechnology (1 mentions)
Calretinin, by Santa Cruz Biotechnology (1 mentions)
Azide fluorescein, by Jena Biosciences (1 mentions)
Mash1, by Santa Cruz Biotechnology (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Doublecortin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using recombinant fgf2
1
Quantitative phospho-VEGFR2 and phospho-FGFR4 analysis
2
Heparin, FGF2, and PF4 Binding
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Heparin from porcine intestinal mucosa, anionic citrate and mesoglycan sulfate were purchased from Sigma. Recombinant FGF2 and PF4 were from R&D, France. Rabbit anti-FGF2 antibodies were from Santa-Cruz Ltd.
3
Molecular Signaling Pathway Analysis
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Lysates were prepared in 30 mmol/L Tris-HCL, pH 7.5, 50 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L NaF, 30 mmol/L NaPPi, 1% Triton, 0.5% IGEPAL, 10% Glycerol, 1 mmol/L Vanadate, 1 mmol/L bpPhen (Calbiochem), and protease inhibitor (Roche) and western blotting and immunoprecipitation were as described [21] (link), [22] (link). Antibodies against FGFR2 included N-terminus MAB6841 (R&D Systems, Minneapolis, MN) and H80 (Santa Cruz), C-terminus C-20 (Santa Cruz) and Y653/654 specific 3471 ((Cell Signaling Technology, (CST), Danvers MA.)). Additional antibodies from CST were: pAKT S473 (4060), AKT (9272), pERK (4370), ERK (9102), pS6RP S235/236 (2217), S6RP (2211), cleaved PARP (9542), beta actin (4967), p105 NFKB S933, p105 NFKB, GAB1 Y672, FRS2 Y436, SHC Y317 CRKII Y221, PRAS40 T246, PDK1 S241, GSKIII S9, PKC S660, RSK S227 (PDK1 site), RSK S359/363 (ERK site), AMPK T172 (LKB site) and GAPDH. 4G10 phosphotyrosine antibody was from Upstate (Charlottesville VA). Recombinant FGF2 was from R&D Systems (Minneapolis MN).
4
LACE Assay for Ligand-Receptor Interactions
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LACE assay was performed as previously described [5 (link),25 (link)]. Briefly, frozen sections were incubated in 0.05% NABH4/PBS for 15 min. After several washes in PBS, sections were incubated in 0.1M glycine at 4°C overnight. Control sections were incubated with Heparitinase I (Seikagaku) before proceeding. Sections were then treated with 1% BSA/TBS solution for 10 min before incubation with 3 μM recombinant Fgf2 (R&D Systems) and 9 μM recombinant human Fgfr1α(IIIc)-Fc (R&D Systems); 20 nM recombinant Fgf10 (R&D Systems) and 20 nM recombinant human Fgfr2β(IIIc)-Fc (R&D Systems); 30nM recombinant Fgf8b (R&D Systems) and 100 nM recombinant human Fgfr3(IIIc) (R&D Systems) at 4°C overnight. Formation of ternary structure was detected by incubation of 1/200 anti-human IgG (Fc-specific) Cy3 (Sigma) in 1% BSA/TBS. n = 5 for wild-type embryos, n = 3 for mutant embryos.
5
Neural Stem Cell Differentiation Assay
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Anti-fibroblast growth factor 2 (FGF2) neutralizing antibody, recombinant FGF2, recombinant epidermal growth factor (EGF; R&D Systems, Minneapolis, MN, USA), Erlotinib (ChemieTek LLC, Indianapolis, IN, USA), recombinant brain-derived neurotrophic factor (BDNF) (Prospec Bio, East Brunswick, NJ, USA), 7,8-dihydroxyflavone (TCI America, Philadelphia, PA, USA), 5-Ethynyl-2-deoxyuridine (EdU), azide-fluorescein (Jena Bioscience, Jena, Germany). Antibodies: Mash1, doublecortin (DCX), Notch3, nestin, calretinin, TrkB, glial fibrillary acidic protein, Skp2 (Santa Cruz Biotech, Dallas, TX, USA), NeuN (Millipore, Billerica, MA, USA), Cy3- and fluorescein isothiocyanate (FITC)-linked secondary antibodies (Jackson Immuno-Research, West Grove, PA, USA).
6
Transgenic Osteoblast and Chondrocyte Cell Lines
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All general laboratory reagents were purchased from Sigma Chemical Co. (Poole, UK) unless otherwise stated. All cells were cultured in α–MEM containing 10% batch-tested FCS (Lonza, Slough, UK), antibiotics (penicillin 50U/ml, streptomycin 50μg/ml) and L-glutamine (5mM). The tetracycline-inducible osteoblastic AT9.2 and chondroblastic DT12.4 cell lines were cultured as previously described19 (link),26 (link) in the presence of 10 μg/ml (AT9.2) or 1μg/ml (DT12.4) tetracycline to inhibit transgene expression, and c-Fos was induced following withdrawal of tetracycline for 48h. DT8.6 cells represent a subclone of ATDC5 chondrocytes constitutively overexpressing c-Fos26 (link) and P1.15, P1.7, 131 P cells are clonal osteosarcoma cell lines derived from c-Fos murine transgenic osteosarcomas12 (link). Primary mouse osteoblasts were obtained from newborn mouse calvariae as described19 (link) and MG63 human osteosarcoma cells were purchased from American Type Culture Collection (ATCC). All cell lines were mycoplasma-negative. Recombinant FGF2 was purchased from R&D Systems (Abingdon, UK). Transfections were performed using Effectene (Qiagen, Crawley, UK) and clones were selected in gentamycin (G418). Lentiviral shRNA vectors for FGFR1 were obtained from MISSION™ shRNA (Sigma). The FGFR inhibitor AZD4547 was purchased from ChemieTek (Indianapolis, IN).
7
Transgenic Osteoblast and Chondrocyte Cell Lines
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