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: Plates were coated with the capturing anti-human KGF-1 monoclonal antibody MAB251 (R&D Systems Inc.) at 1.0 μg/ml, washed, and blocked (2% bovine serum albumin [BSA], 5% sucrose, 0.05% NaN 3 ).

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: Plates were coated with the capturing anti-human KGF-1 monoclonal antibody MAB251 (R&D Systems Inc.) at 1.0 μg/ml, washed, and blocked (2% bovine serum albumin [BSA], 5% sucrose, 0.05% NaN 3 ).

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: Plates were coated with the capturing anti-human KGF-1 monoclonal antibody MAB251 (R&D Systems Inc.) at 1.0 μg/ml, washed, and blocked (2% bovine serum albumin [BSA], 5% sucrose, 0.05% NaN 3 ).

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: Plates were coated with the capturing anti-human KGF-1 monoclonal antibody MAB251 (R&D Systems Inc.) at 1.0 μg/ml, washed, and blocked (2% bovine serum albumin [BSA], 5% sucrose, 0.05% NaN 3 ).

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

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