Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Scheme for the construction of the expression plasmids and the structures of their translation products. cDNAs for rat EGF and human bFGF were amplified by PCR from first-strand cDNAs prepared from the poly(A)+ RNA fraction of rat submaxillary gland and a human osteosarcoma cell line, respectively, using primers tagged with extra 5′ restriction sites. Each amplified cDNA was digested with restriction enzymes and then inserted into the BamHI–EcoRI site of pCHC302, giving pCHC302-EGF and pCHC302-bFGF, which express fusion proteins between GST and collagen-binding EGF (GST-CBEGF) and collagen-binding bFGF (GST-CBFGF), respectively. In the structure of GST-CBEGF (GST-CBFGF), amino acid residues derived from the pGEX-4T-2 plasmid vector are given in the single-letter code, and the thrombin-cleavage site is indicated by an arrow. The numbers in parentheses are the molecular weights of the domains.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Expressing, Amplification, Binding Assay, Derivative Assay, Plasmid Preparation

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Purification profiles of CBEGF and CBFGF on SDS/PAGE. Samples at each purification step for CBEGF (lanes 2–5) and CBFGF (lanes 6–9), and the final preparation of human recombinant bFGF (lane 10) were electrophoretically separated in a SDS/13% polyacrylamide gel under reducing conditions and then stained with Coomassie brilliant blue R-250. Lane 1, molecular weight markers; lane 2, E. coli BL21/pCHC302-EGF crude extract; lanes 3 and 7, eluate from glutathione-Sepharose; lanes 4 and 8, thrombin digest of the eluate from glutathione-Sepharose; lane 5, eluate from Resource Q (purified CBEGF); lane 6, E. coli BL21/pCHC302-bFGF crude extract; lane 9, eluate from heparin-Sepharose (purified CBFGF); lane 10, purified human recombinant bFGF.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Purification, SDS Page, Recombinant, Staining, Molecular Weight

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Dose–response curves for the growth factor activity of rat EGF, CBEGF, human bFGF, and CBFGF. BALB/c 3T3 A31 cells (2 × 104 cells in 2 ml of DMEM-2% calf serum) were inoculated onto 6-well multiwell plates, and 7 h later test samples were added. The cell number was determined with a Coulter counter after 4 days culture. The cell numbers in the absence of test samples and in the presence of 10% calf serum were 29,100 ± 1,600 and 239,600 ± 9,900, respectively. Each point represents the mean value ± SEM for triplicate experiments.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Activity Assay

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Detection of S-phase cells as BrdU incorporation and immunohistochemical staining of CBFGF in subcutaneous tissue of nude mice injected with CBFGF and human bFGF. The animals received an i.p. injection of BrdU (10 mg/100 g body weight) 24 h before death. Immunolocalization was performed on 5-μm paraffin sections by the streptoavidin-biotin-alkaline phosphatase complex technique using anti-BrdU mAbs (a-d) or anti-human bFGF antibodies (e and f) as the primary antibodies. (a, e, and f) 5 days after injection of CBFGF (50 μg). (b) 7 days after injection of CBFGF. (c and d) 5 days and 7 days, respectively, after injection of human bFGF (20 μg). (Bars, 100 μm.)

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: BrdU Incorporation Assay, Immunohistochemical staining, Staining, Injection

Journal: eLife

Article Title: FGF2-FGFR1 signaling regulates release of Leukemia-Protective exosomes from bone marrow stromal cells

doi: 10.7554/eLife.40033

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit polyclonal anti-FGF2 , Santa Cruz , Sc-79 , Dilution 1:500.

Techniques: shRNA, Plasmid Preparation, CRISPR, Recombinant, Transfection, Software

Journal: PLoS ONE

Article Title: Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone

doi: 10.1371/journal.pone.0014033

Figure Lengend Snippet: Endogenous levels of FGF-2 were analyzed on FOb and POb cells by immunoblotting using antiFGF-2 antibody. Higher levels of high molecular weight (HMW) and low molecular weight (LMW) FGF-2 forms were observed in FOb compared to POb at all stages analyzed. The total cellular FGF-2 proteins were quantified by densitometry (lower panel), α-tubulin was used as loading control. Histogram represents the densitometric analysis of electrophoresis bands, the relative intensities of bands were normalized to their respective loading control and set as 100% The results are presented as the mean ± SD of three independent experiments. The asterisks represent P value <0.001.

Article Snippet: A polyclonal goat anti-FGF-2 antibody (R&D systems, MN) was used for FGF-2 neutralization.

Techniques: Western Blot, Molecular Weight, Electrophoresis

Journal: PLoS ONE

Article Title: Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone

doi: 10.1371/journal.pone.0014033

Figure Lengend Snippet: Cell proliferation of FOb and POb harvested from E17.5 mice was measured by BrdU. Osteoblast cells were cultured with conditioned serum-free media collected from frontal osteoblasts (FCM) or parietal osteoblasts (PCM) after 24 hours culture. The conditioned media were incubated with neutralizing anti-FGF-2 antibody at different concentrations (1, 2 and 4 µg/ml). Same concentrations of irrelevant rabbit IgG were used as control. 20 ng/ml of hrFGF-2 was added to the medium as a positive control (SF + FGF-2) and serum-free medium as a negative control (SF). FCM induced significant mitogenic effect, as much as the exogenous added rhFGF-2, on both FOb and POB cells. Anti-FGF-2 antibody blocked the FGF-2 mitogenic effect in a dose-dependent manner. Conversely PCM did not elicit mitogenic effect either on FOb or POb cells and there was no blocking effect by the FGF-2 neutralizing antibody on PCM.

Article Snippet: A polyclonal goat anti-FGF-2 antibody (R&D systems, MN) was used for FGF-2 neutralization.

Techniques: Cell Culture, Incubation, Positive Control, Negative Control, Blocking Assay

Journal: PLoS ONE

Article Title: Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone

doi: 10.1371/journal.pone.0014033

Figure Lengend Snippet: FOb and POb cells harvested from E17.5 mice were cultured in serum free α-MEM for 12 hours in presence of specific inhibitors of the FGF signaling pathways to pre-empty endogenous FGF-2 activity. Then cells were treated with 20 ng/ml of rhFGF-2. A , effect of exogenous FGF-2 and inhibitors of MAPK signaling pathway. FGF-2 stimulation for 30 minutes increased phosphorylation of ERK protein equally in FOb and POb cells. Co-treatment with 10 µM U0126 inhibitor inhibited the FGF-2 induction as well as the endogenous phosphorylation of pERK protein in untreated FGF-2 FOb and POb cells. Histogram represents densitometric analysis of electrophoresis bands as above. B , effect of exogenous FGF-2 and inhibitors of PI3K signaling pathway. FGF-2 treatment resulted in increased pAkt phosphorylation which was two fold higher in POb cells compared to FOb cells. Treatment with 20 µM LY294002 inhibitor mostly abrogated the effect of exogenous FGF-2 and drastically reduced the endogenous level of pAkt protein in untreated FGF-2 FOb cells. Histogram represents densitometric analysis of electrophoresis bands. C , effect of exogenous FGF-2 and inhibitors of PKC α/β signaling pathway. FGF-2 treatment also induced an increased phosphorylation of PKC α/β proteins which was inhibited by co-treatment with 2 µM GÖ6983 inhibitor. Treatment with the inhibitor also reduced the endogenous level of p PKC α/β proteins in untreated FGF-2 cells. Histogram represents densitometric analysis of electrophoresis bands. D , effect of exogenous FGF-2 and inhibitors of PKC δ signaling pathway. FGF-2 and/or inhibitor treatments on PKC δ protein produced a similar effect to that observed on PKC α/β proteins. Asterisk * represents statistical significance (*p<0.05).

Article Snippet: A polyclonal goat anti-FGF-2 antibody (R&D systems, MN) was used for FGF-2 neutralization.

Techniques: Cell Culture, Activity Assay, Electrophoresis, Produced