Journal:

Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4

doi: 10.1128/IAI.70.12.6541-6548.2002

Figure Lengend Snippet: LPS purified from E. coli significantly induces gingival fibroblast expression of KGF-1 protein in the presence or absence of serum. Gingival fibroblasts were cultured to quiescence in α-MEM supplemented with 1% FBS. Various concentrations of LPS (A) and recombinant human sCD14 (B), with or without LPS (50 ng/ml), were added to serum-free and 1% FBS-supplemented α-MEM. A sandwich ELISA was used to analyze KGF-1 protein levels in 24-h conditioned medium. Mean ± standard deviation; n = 4.

Article Snippet: Wells were washed, and 100 μl of biotinylated anti-human KGF-1 polyclonal antibody (BAF251; 200 ng/ml; R&D Systems Inc.) was added, the wells were incubated for 2 h, and then the wells were washed.

Techniques: Purification, Expressing, Cell Culture, Recombinant, Sandwich ELISA, Standard Deviation

Journal:

Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4

doi: 10.1128/IAI.70.12.6541-6548.2002

Figure Lengend Snippet: Addition of CD14-blocking antibody inhibits LPS stimulation of KGF-1 protein expression. Gingival fibroblasts were brought to quiescence in α-MEM supplemented with 1% FBS. In replicate wells, the following treatment groups were tested: α-MEM with 1% FBS (control), α-MEM with 1% FBS plus 50 ng of E. coli LPS (LPS) per ml or first pretreatment for 30 min with 0.5 μg of anti-CD14 blocking antibody per ml with α-MEM and 1% FBS prior to the addition of 50 ng of LPS per ml. KGF-1 protein in the 24-h conditioned medium was measured by a sandwich ELISA. Mean ± standard deviation; n = 4.

Article Snippet: Wells were washed, and 100 μl of biotinylated anti-human KGF-1 polyclonal antibody (BAF251; 200 ng/ml; R&D Systems Inc.) was added, the wells were incubated for 2 h, and then the wells were washed.

Techniques: Blocking Assay, Expressing, Sandwich ELISA, Standard Deviation

Journal:

Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4

doi: 10.1128/IAI.70.12.6541-6548.2002

Figure Lengend Snippet: LPS downregulates cell membrane expression of TLR2 and TLR4. Gingival fibroblasts were treated with either E. coli or P. gingivalis (P. ging) LPS (50 ng/ml), collected at various times, fixed, stained without permeabilization with either anti-TLR2 (TLR2) or anti-TLR4 (TLR4) monoclonal antibody, and then stained with an alexa 488 nm-conjugated secondary antibody prior to flow cytometry analysis. Mean ± standard deviation; n = 4. (B) Preincubation of cultures with TLR2 and TLR4 blocking antibodies inhibited E. coli LPS stimulation of KGF-1 protein expression. Culture groups were treated with α-MEM with 1% FBS (control) or α-MEM with 1% FBS and 50 ng of LPS (LPS) per ml or first pretreated for 30 min with either 0.5 μg of anti-TLR2 (LPS+aT2) or TLR4 (LPS+aT4) blocking antibody per ml with α-MEM plus 1% FBS prior to the addition of LPS at 50 ng/ml. A sandwich ELISA was used to measure KGF-1 protein expression in 24-h conditioned medium. Mean ± standard deviation; n = 4.

Article Snippet: Wells were washed, and 100 μl of biotinylated anti-human KGF-1 polyclonal antibody (BAF251; 200 ng/ml; R&D Systems Inc.) was added, the wells were incubated for 2 h, and then the wells were washed.

Techniques: Membrane, Expressing, Staining, Flow Cytometry, Standard Deviation, Blocking Assay, Sandwich ELISA

Journal:

Article Title: Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4

doi: 10.1128/IAI.70.12.6541-6548.2002

Figure Lengend Snippet: LPS induction of KGF-1 protein expression is regulated by the transcription factors AP-1 and NF-κB. Quiescent gingival fibroblasts in α-MEM with 1% FBS were preincubated for 30 min with increasing concentrations of the transcription factor inhibitors curcumin (A) and PDTC (B) prior to the addition of E. coli LPS (50 μg/ml). Conditioned medium was collected at 24 h, and the KGF-1 protein level was assayed by a sandwich ELISA. The dotted line represents the KGF-1 protein expression of the negative control (1% serum alone). RNA from cultures was isolated at 6 h for Northern analysis (C). Lanes: 1, negative control (1% serum alone); 2, E. coli LPS at 50 ng/ml; 3, LPS plus PDTC at 2 nM; 4, LPS plus PDTC at 4 nM; 5, LPS plus PDTC at 6 nM; 6, LPS plus curcumin at 5 μM; 7, LPS plus curcumin at 10 μM; 8, LPS plus curcumin at 30 μM.

Article Snippet: Wells were washed, and 100 μl of biotinylated anti-human KGF-1 polyclonal antibody (BAF251; 200 ng/ml; R&D Systems Inc.) was added, the wells were incubated for 2 h, and then the wells were washed.

Techniques: Expressing, Sandwich ELISA, Negative Control, Isolation, Northern Blot

Journal: Cellular & molecular immunology

Article Title: Umbilical cord-derived mesenchymal stem cells regulate thymic epithelial cell development and function in Foxn1(-/-) mice.

doi: 10.1038/cmi.2013.69

Figure Lengend Snippet: Figure 7 UC-MSCs can engraft into the thymus and express several proteins, which may enhance thymic development. (a) CM-Dil-labeled UC- MSCs migrated and engrafted into the thymi and were found primarily at the thymic rim. (b) Flow and immunofluorescence analysis revealed that UC-MSCs expressed CD248, which may be required for optimal post-natal thymic growth and regeneration following infection-dependent thymic atrophy. (c) RT-PCR analysis of the different cytokine mRNA expression levels in UC-MSCs; MSCs express high levels of ghrelin, KGF, GHR and IL- 15, and low levels of GH and IL-7. (d) Frozen thymic sections stained with DAPI (blue) and anti-human KGF (green). Foxn12/2 mice were injected with UC-MSCs intraperitoneally, 23106 cells/mice. After 72 h, thymi were collected and cut into frozen sections. DAPI, 2-(4-Amidinophenyl)-6- indolecarbamidine dihydrochloride; KGF, keratinocyte growth factor; GH, growth hormone; GHR, growth hormone receptor; GHN, ghrelin; US- MSC, umbilical cord-derived mesenchymal stem cell.

Article Snippet: After 24 h, cells and frozen sections of the thymic tissues were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 3% normal donkey serum, and were then incubated overnight at 4 uC with goat anti-human KGF/ FGF7 antibody (R&D Systems, Minneapolis, MN, USA) at 10 mg/ml.

Techniques: Labeling, Immunofluorescence, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Injection, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7.

doi: 10.1074/jbc.275.10.7095

Figure Lengend Snippet: FIG. 1. Perlecan binds to FGF7 in overlay assays. A, binding of soluble recombinant FGF7 (100 ng/ml) to immobilized perlecan purified from either human colon carcinoma (WiDr) or human umbilical artery endothelial cells (EC); type I collagen or DMEM served as negative controls. Proteins were slot-blotted unto nitrocellulose filters at the indicated concentrations, blocked in 5% milk, and incubated with sol- uble ligand. Detection was with a specific anti-FGF7 monoclonal anti- body and chemiluminescence. B, binding of soluble human EC perlecan (5 mg/ml) to immobilized FGF7 or DMEM. The reaction was detected with specific monoclonal antibody against perlecan domain III. C, bind- ing of soluble FGF7 to perlecan preparation treated with heparitinase I alone or following a sequential digestion with heparitinase I and plas- min, thrombin, or collagenase. The products were separated on an 8% SDS-PAGE, transferred onto nitrocellulose, and overlaid with soluble FGF7. Specific binding was detected with anti-FGF7 antibody. The migration of molecular mass markers is indicated in the left margin. D, the blot in C was stripped and reacted with a monoclonal antibody against domain III of perlecan. Notice the presence of domain III- specific epitopes in the ;50-kDa plasmin digest (double arrowheads, lane 2) and in the ;30-kDa thrombin-generated fragment (arrow, lane 3).

Article Snippet: Antibodies used in this study include: the C19 monoclonal against FGF7 (Santa Cruz Biotechnology), an affinity-purified goat polyclonal antibody raised against the C terminus residues 176–194, and monoclonal antibodies against either domain III (15) or other domains of human perlecan (13).

Techniques: Binding Assay, Recombinant, Purification, Incubation, SDS Page, Migration, Generated

Journal: The Journal of biological chemistry

Article Title: The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7.

doi: 10.1074/jbc.275.10.7095

Figure Lengend Snippet: FIG. 2. Binding of various domains of perlecan to recombinant FGF7. A, schematic representation of the five domains of human per- lecan indicated by Roman numerals. Each domain is color-coded. The number of the amino acid encoding the various constructs is shown in the bottom. B, SDS-PAGE of various domains following purification as MBP-fusion proteins. The migration of protein standard is indicated on the left margins. C, affinity of 125I-FGF7 to various domains of human perlecan. About 10 pmol of each domain was bound to Immulon® wells in triplicate and incubated with equal amounts of 125I-FGF7 (1–2 3 1018

Article Snippet: Antibodies used in this study include: the C19 monoclonal against FGF7 (Santa Cruz Biotechnology), an affinity-purified goat polyclonal antibody raised against the C terminus residues 176–194, and monoclonal antibodies against either domain III (15) or other domains of human perlecan (13).

Techniques: Binding Assay, Recombinant, Construct, SDS Page, Purification, Migration, Incubation

Journal: The Journal of biological chemistry

Article Title: The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7.

doi: 10.1074/jbc.275.10.7095

Figure Lengend Snippet: FIG. 3. Saturation curves (A and C) and Scatchard plots (B and D) for the binding of perlecan domain III (G), MBP (E), or per- lecan domain V (f) to iodinated FGF7. 125I-FGF7 (1–2 3 1018

Article Snippet: Antibodies used in this study include: the C19 monoclonal against FGF7 (Santa Cruz Biotechnology), an affinity-purified goat polyclonal antibody raised against the C terminus residues 176–194, and monoclonal antibodies against either domain III (15) or other domains of human perlecan (13).

Techniques: Binding Assay

Journal: The Journal of biological chemistry

Article Title: The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7.

doi: 10.1074/jbc.275.10.7095

Figure Lengend Snippet: FIG. 4. In vivo interaction between FGF7 and perlecan protein core. A, schematic representation of domain III construct and various deletion mutants. The numbers on the right designate the amino acid position based on the mature protein core. The growth is also indicated by semiquantitative assessment with maximal growth at 111. B, representative growth of three individual clones carrying the N-terminal half of domain III (III-D2). The empty areas include negative pDT1 and pLAM59controls. C, schematic representation of domain IV and its growth status. D, schematic representation of domain V and its growth status. E, b-galactosidase assays using various domains of perlecan (left row) or FGF7 (right row) as bait or prey, respectively. The picture shows some of the blue colonies obtained after the induction of the b-galactosidase activity due to the interaction of the analyzed proteins. The plasmids pTD1 combined with pLAM59 and p53 (harboring the p53 gene) combined with pSV40 (harboring the SV40 large T antigen gene) were used as negative and positive controls, respectively. F, b-galactosidase assays using various perlecan domains as indicated. The positive b-galactosidase activity was also supported by growth in triple minus media (not shown).

Article Snippet: Antibodies used in this study include: the C19 monoclonal against FGF7 (Santa Cruz Biotechnology), an affinity-purified goat polyclonal antibody raised against the C terminus residues 176–194, and monoclonal antibodies against either domain III (15) or other domains of human perlecan (13).

Techniques: In Vivo, Construct, Clone Assay, Activity Assay