napi-iib antibody  (Santa Cruz Biotechnology)

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Effect of inflammation on intestinal phosphate absorption and type IIb sodium-phosphate cotransporter (NaPi-IIb) expression in trinitrobenzene sulfonic acid (TNBS) colitis mice. Six-week-old male mice were treated with TNBS (2 mg/mouse) and were harvested 6 days after TNBS administration. A: brush-border membrane (BBM) phosphate uptake in TNBS colitis mice. BBM vesicles (BBMVs) were isolated from the ileal mucosa of mice treated with PBS or TNBS, and BBM phosphate uptake was performed. The contribution of sodium-dependent uptake was calculated by subtracting the sodium-independent uptake values observed in the absence of sodium from the uptake values in the presence of sodium. Data presented are means ± SE in 3 or 4 different groups of animals. *P ≤ 0.028 for TNBS mice vs. control mice. B: NaPi-IIb expression in TNBS colitis mice. BBMVs were isolated from the ileal mucosa of mice treated with PBS or TNBS and used for Western detection. A 1:4,000 dilution of the mouse NaPi-IIb antibody was used. The expression of NaPi-IIb protein is calculated by the density of NaPi-IIb band over that of β-actin band. Bar chart shows NaPi-IIb protein expression indicated as means ± SE in the sum of 3 independent experiments. *P ≤ 0.017 for control groups vs. TNBS groups. Inset: typical immunoblot image.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Isolation, Western Blot

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Effect of inflammation on NaPi-IIb expression in TNBS colitis rats. Three-week-old rats were treated with TNBS (1 mg/rat) and were harvested 6 days after TNBS administration. A: BBMVs were isolated from the jejunal mucosa of rats treated with PBS or TNBS and used for Western detection. A 1:4,000 dilution of the mouse NaPi-IIb antibody was used. The expression of NaPi-IIb protein is calculated by the density of NaPi-IIb band over that of β-actin band. Bar chart shows NaPi-IIb protein expression indicated as means ± SE in the sum of 3 independent experiments. *P ≤ 0.04 for control groups vs. TNBS groups. Inset: a typical immunoblot image. B: RNAs were isolated from the jejunal mucosa of control or TNBS rats and used for real-time PCR. NaPi-IIb mRNA and TATA binding protein (TBP) mRNA were amplified with rat-specific NaPi-IIb and TBP primers. The changes in NaPi-IIb gene expression are analyzed by the comparative cycle threshold (Ct) method. Data are means ± SE from total 18 rats (9 for TNBS group, 9 for control group). *P ≤ 0.004 for control group vs. TNBS group.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Isolation, Western Blot, Real-time Polymerase Chain Reaction, Binding Assay, Amplification

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Effect of TNF-α on phosphate absorption and NaPi-IIb expression in Caco-2 cells. A: phosphate uptake in Caco-2 cells. Cellular phosphate uptake was performed from control and TNF-α-treated (20 ng/ml, 40 h) cells. The contribution of sodium-dependent uptake was calculated by subtracting the sodium-independent uptake values observed in the absence of sodium from the uptake values in the presence of sodium. Data presented are means ± SE from 3 separate experiments. *P ≤ 0.013 for TNF-α treatment vs. control. B: NaPi-IIb protein expression in Caco-2 cells. Crude membrane protein was prepared from control and TNF-α-treated (20 ng/ml, 40 h) cells. Western blot was performed, and human NaPi-IIb protein was detected by an anti-human sodium-phosphate cotransporter antibody. Data are means ± SE from 3 separate experiments. *P ≤ 0.006 for TNF-α treatment vs. control. Inset: typical immunoblot image. C: NaPi-IIb mRNA expression in Caco-2 cells. RNA was isolated from control and TNF-α-treated (20 ng/ml, 40 h) cells and used for first-strand cDNA synthesis. Real-time PCR was performed with human NaPi-IIb or TBP primers in separate reactions. Results are means ± SE from 4 separate experiments. *P ≤ 0.001 for control vs. TNF-α treatment.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: TNF-α response region on human NaPi-IIb gene promoter. A: Caco-2 cells were cotransfected with human NaPi-IIb promoter constructs (pGL3/-2783, pGL3/-1103, pGL3/-380, pGL3/-58) and pRL-CMV. TNF-α (20 ng/ml) was applied 40 h before measuring promoter activities. The degree of inhibition is shown as the ratio of luciferase activity in TNF-α-treated cells over luciferase activity in vehicle-treated cells. Results are means ± SE from 6 separate experiments. *P < 0.01 for control vs. TNF-α treatment. B: identification of nuclear protein bound on promoter region (−37 bp/−13 bp) by gel mobility shift assays (GMSAs). A 32P-labeled double-stranded oligonucleotide probe covering the proximal promoter region (−37 bp/−13 bp) was incubated with 5 μg of Caco-2 cell nuclear extract in the presence or absence of unlabeled 100 × excess NF1 consensus oligos (NF1 oligos), 4 μg rabbit IgG or anti-Elk antibody (αELK) or anti-NF1 antibody (αNF1). Image is representative of 3 independent experiments. C: identification of DNA region involving TNF-α regulation. Nuclear proteins were isolated from Caco-2 cells treated with normal medium or TNF-α medium. GMSAs were performed with DNA probe covering the basal promoter region (−37 bp/−13 bp). Results shown are representative of 4 separate experiments.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Construct, Inhibition, Luciferase, Activity Assay, Mobility Shift, Labeling, Incubation, Isolation

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Signaling pathways of TNF-α effect on NaPi-IIb expression. Caco-2 cells were cotransfected with human NaPi-IIb promoter construct (pGL3/−58) and pRL-CMV. Various inhibitors were applied 2 h before TNF-α treatment (20 ng/ml, 40 h). The degree of inhibition is shown as the ratio of luciferase activity in TNF-α-treated cells over luciferase activity in vehicle-treated cells. Results are means ± SE from 6 separate experiments. *P ≤ 0.01 for control vs. TNF-α treatment. EGFR, EGF receptor. PD, PD098059; AG, AG1478.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Construct, Inhibition, Luciferase, Activity Assay

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Effect of inflammation on intestinal phosphate absorption and type IIb sodium-phosphate cotransporter (NaPi-IIb) expression in trinitrobenzene sulfonic acid (TNBS) colitis mice. Six-week-old male mice were treated with TNBS (2 mg/mouse) and were harvested 6 days after TNBS administration. A: brush-border membrane (BBM) phosphate uptake in TNBS colitis mice. BBM vesicles (BBMVs) were isolated from the ileal mucosa of mice treated with PBS or TNBS, and BBM phosphate uptake was performed. The contribution of sodium-dependent uptake was calculated by subtracting the sodium-independent uptake values observed in the absence of sodium from the uptake values in the presence of sodium. Data presented are means ± SE in 3 or 4 different groups of animals. *P ≤ 0.028 for TNBS mice vs. control mice. B: NaPi-IIb expression in TNBS colitis mice. BBMVs were isolated from the ileal mucosa of mice treated with PBS or TNBS and used for Western detection. A 1:4,000 dilution of the mouse NaPi-IIb antibody was used. The expression of NaPi-IIb protein is calculated by the density of NaPi-IIb band over that of β-actin band. Bar chart shows NaPi-IIb protein expression indicated as means ± SE in the sum of 3 independent experiments. *P ≤ 0.017 for control groups vs. TNBS groups. Inset: typical immunoblot image.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Isolation, Western Blot

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Effect of inflammation on NaPi-IIb expression in TNBS colitis rats. Three-week-old rats were treated with TNBS (1 mg/rat) and were harvested 6 days after TNBS administration. A: BBMVs were isolated from the jejunal mucosa of rats treated with PBS or TNBS and used for Western detection. A 1:4,000 dilution of the mouse NaPi-IIb antibody was used. The expression of NaPi-IIb protein is calculated by the density of NaPi-IIb band over that of β-actin band. Bar chart shows NaPi-IIb protein expression indicated as means ± SE in the sum of 3 independent experiments. *P ≤ 0.04 for control groups vs. TNBS groups. Inset: a typical immunoblot image. B: RNAs were isolated from the jejunal mucosa of control or TNBS rats and used for real-time PCR. NaPi-IIb mRNA and TATA binding protein (TBP) mRNA were amplified with rat-specific NaPi-IIb and TBP primers. The changes in NaPi-IIb gene expression are analyzed by the comparative cycle threshold (Ct) method. Data are means ± SE from total 18 rats (9 for TNBS group, 9 for control group). *P ≤ 0.004 for control group vs. TNBS group.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Isolation, Western Blot, Real-time Polymerase Chain Reaction, Binding Assay, Amplification

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Effect of TNF-α on phosphate absorption and NaPi-IIb expression in Caco-2 cells. A: phosphate uptake in Caco-2 cells. Cellular phosphate uptake was performed from control and TNF-α-treated (20 ng/ml, 40 h) cells. The contribution of sodium-dependent uptake was calculated by subtracting the sodium-independent uptake values observed in the absence of sodium from the uptake values in the presence of sodium. Data presented are means ± SE from 3 separate experiments. *P ≤ 0.013 for TNF-α treatment vs. control. B: NaPi-IIb protein expression in Caco-2 cells. Crude membrane protein was prepared from control and TNF-α-treated (20 ng/ml, 40 h) cells. Western blot was performed, and human NaPi-IIb protein was detected by an anti-human sodium-phosphate cotransporter antibody. Data are means ± SE from 3 separate experiments. *P ≤ 0.006 for TNF-α treatment vs. control. Inset: typical immunoblot image. C: NaPi-IIb mRNA expression in Caco-2 cells. RNA was isolated from control and TNF-α-treated (20 ng/ml, 40 h) cells and used for first-strand cDNA synthesis. Real-time PCR was performed with human NaPi-IIb or TBP primers in separate reactions. Results are means ± SE from 4 separate experiments. *P ≤ 0.001 for control vs. TNF-α treatment.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: TNF-α response region on human NaPi-IIb gene promoter. A: Caco-2 cells were cotransfected with human NaPi-IIb promoter constructs (pGL3/-2783, pGL3/-1103, pGL3/-380, pGL3/-58) and pRL-CMV. TNF-α (20 ng/ml) was applied 40 h before measuring promoter activities. The degree of inhibition is shown as the ratio of luciferase activity in TNF-α-treated cells over luciferase activity in vehicle-treated cells. Results are means ± SE from 6 separate experiments. *P < 0.01 for control vs. TNF-α treatment. B: identification of nuclear protein bound on promoter region (−37 bp/−13 bp) by gel mobility shift assays (GMSAs). A 32P-labeled double-stranded oligonucleotide probe covering the proximal promoter region (−37 bp/−13 bp) was incubated with 5 μg of Caco-2 cell nuclear extract in the presence or absence of unlabeled 100 × excess NF1 consensus oligos (NF1 oligos), 4 μg rabbit IgG or anti-Elk antibody (αELK) or anti-NF1 antibody (αNF1). Image is representative of 3 independent experiments. C: identification of DNA region involving TNF-α regulation. Nuclear proteins were isolated from Caco-2 cells treated with normal medium or TNF-α medium. GMSAs were performed with DNA probe covering the basal promoter region (−37 bp/−13 bp). Results shown are representative of 4 separate experiments.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Construct, Inhibition, Luciferase, Activity Assay, Mobility Shift, Labeling, Incubation, Isolation

Journal:

Article Title: Tumor necrosis factor-alpha impairs intestinal phosphate absorption in colitis

doi: 10.1152/ajpgi.90722.2008

Figure Lengend Snippet: Signaling pathways of TNF-α effect on NaPi-IIb expression. Caco-2 cells were cotransfected with human NaPi-IIb promoter construct (pGL3/−58) and pRL-CMV. Various inhibitors were applied 2 h before TNF-α treatment (20 ng/ml, 40 h). The degree of inhibition is shown as the ratio of luciferase activity in TNF-α-treated cells over luciferase activity in vehicle-treated cells. Results are means ± SE from 6 separate experiments. *P ≤ 0.01 for control vs. TNF-α treatment. EGFR, EGF receptor. PD, PD098059; AG, AG1478.

Article Snippet: A 1:4,000 dilution of mouse NaPi-IIb antibody ( 42 ) or 1:1,000 dilution of human NaPi-IIb antibody (Alpha Diagnostic International, San Antonio, TX) was used to detect NaPi-IIb protein.

Techniques: Expressing, Construct, Inhibition, Luciferase, Activity Assay