Journal: Poultry science

Article Title: Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

doi: 10.3382/ps/pev085

Figure Lengend Snippet: Figure 1. Location of 8 selected peptide regions on the human intestinal sodium-dependent phosphate co-transporter (NaPi2b). All selected peptides are located on the extracellular, intestinal lumen side, which is accessible to dietary phosphate. Black cylinders indicate 8 transmembrane domains, with 4 extracellular and 3 intracellular loops. See Table 1 for specific human and mouse peptide sequences. Abbreviations: ECL = extracellular lumen, ICL = intracellular lumen. Human sequence Uniprot ID: O95436. Figure adapted from Cook et al., U.S. Patent Application 20100166760.

Article Snippet: Anti-m16 antibody and commercial NaPi2b antibody (positive control; sc-33928; goat polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA) were incubated on slides overnight at 1:100 dilution in normal antibody diluent (Fisher Scientific, Pittsburgh, PA).

Techniques: Sequencing

Journal: Poultry science

Article Title: Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

doi: 10.3382/ps/pev085

Figure Lengend Snippet: Figure 2. A and B. The ability of antibodies against sodium- dependent phosphate co-transporter 2b (NaPi2b) peptides to in- hibit sodium dependent radiolabeled phosphate uptake by NaPi2b- expressing Caco-2 cells. Experiments were conducted in triplicate and data points represent mean±SEM. A. Caco-2 cells were incubated with nicotinamide (100 μM, a known NaPi2b inhibitor), control non- specific antibodies, or peptide-specific anti-NaPi2b antibodies 12 to 19 (0.1 mg/mL) for 1 h. Cells were washed and then incubated with 32PO4 in the presence and absence of sodium for 20 min. Cell uptake of 32PO4 was determined. Net activity was the difference in transport in the presence and absence of sodium (measured in cpm; an aster- isk denotes P ≤0.05 versus nicotinamide). Reductions in transport are shown as percent of nonspecific control, which remained at 100% transport postantibody incubation (i.e., no reduction in transport). B. Antibodies at different dilutions were used to determine the optimal concentration for phosphate uptake inhibition studies. Starting with a concentration 500 μg/mL, antibodies were prepared in a series of 5-fold dilutions. Only the results for Antibody 16 are shown; however, all an- tibodies showed similar profiles and exhibited optimal activity at the 100 μg/mL dilution. The ability of each dilution to inhibit phosphate uptake was measured as described.

Article Snippet: Anti-m16 antibody and commercial NaPi2b antibody (positive control; sc-33928; goat polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA) were incubated on slides overnight at 1:100 dilution in normal antibody diluent (Fisher Scientific, Pittsburgh, PA).

Techniques: Expressing, Incubation, Control, Activity Assay, Concentration Assay, Inhibition

Journal: Poultry science

Article Title: Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

doi: 10.3382/ps/pev085

Figure Lengend Snippet: Figure 3. Specificity of anti-h16 (human, h16 and mouse, m16, homologs) antibodies for peptides h16 and m16 found on the sodium phosphate co-transporter 2b (2b). Peptide h16 (TSPSLCWT) or Pep- tide m16 (TSPSYCWT) were conjugated to ovalbumin and coated on a 96 well plate for use in an ELISA. Four dilutions of antibody ex- tracted from egg yolk of hens producing control, anti-h16, or anti-m16 were incubated with each coating. Binding of the primary egg antibody to each of the coatings was visualized using horseradish peroxidase (HRP) enzyme-linked secondary antibody and substrate (reported as optical density, see Methods section). Lines for control on h16 and m16 are overlapping each other. Data show that control antibody does not bind either antibody, but antibody to peptides h16 or m16 cross- reacts with each peptide. The term h16 refers to the human sequence and m16 to the mouse sequence of peptide 16. Dotted lines indicate peptide m16 coating, and solid lines indicate h16 coating. For example, h16 on m16 = antibody made to NaPi2b human peptide sequence was reacted with mouse peptide 16 (m16) conjugated to OVA and coated on the plate.

Article Snippet: Anti-m16 antibody and commercial NaPi2b antibody (positive control; sc-33928; goat polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA) were incubated on slides overnight at 1:100 dilution in normal antibody diluent (Fisher Scientific, Pittsburgh, PA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Incubation, Binding Assay, Sequencing

Journal: Poultry science

Article Title: Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

doi: 10.3382/ps/pev085

Figure Lengend Snippet: Figure 5. Anti-m16 antibody binds NaPi2b in mouse ileum in the same region as commercial NaPi2b antibody. Ileum from C57Bl6/J mice that were not fed egg antibody were collected, fixed in forma- lin, and paraffin sectioned. Commercial antibody was stained on 2 slides (Figure 5A, 5B) and either an adjuvant injected control IgY anti- body (Figure 5C) or the anti-m16 antibody (Figure 5D) was incubated on separate slides to determine if anti-m16 and the commercial anti- NaPi2b antibody interacted in the same place. We demonstrate that the commercial antibody and only anti-m16 interact in the same place on a murine ileal villus, as shown by the yellow color when the slides were overlaid (400× magnification in all slides, Figure 5F), whereas control does not (Figure 5E). These results provide evidence that our antibody was successfully made and is specific for murine NaPi2b.

Article Snippet: Anti-m16 antibody and commercial NaPi2b antibody (positive control; sc-33928; goat polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA) were incubated on slides overnight at 1:100 dilution in normal antibody diluent (Fisher Scientific, Pittsburgh, PA).

Techniques: Staining, Adjuvant, Injection, Control, Incubation

Journal: Poultry science

Article Title: Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

doi: 10.3382/ps/pev085

Figure Lengend Snippet: Figure 6. Mean serum phosphate ± SEM of mice orally supple- mented with yolk powder containing antibody to Peptide 16 on the sodium phosphate cotransporter 2b (anti-m16 or anti-h16, 1% of diet) or sevelamer (1% of the diet) after 14 d supplementation (n = 21/treat- ment). Serum phosphate was not affected by either supplement at d 0 or 7 but at d 14 sevelamer and each anti-NaPi2b antibody (where h16 = human peptide sequence and m16 = mouse peptide sequence) decreased serum phosphate when compared to mice fed the control antibody. Versus control; ∗∗Control+Sev, P < 0.0001, anti-m16, ∗P = 0.019, anti-h16, P = 0.066. Sevelamer vs. anti-m16, P = 0.07. BW was unaffected by the supplement treatments: average BW at d 0, 23.49 ± 0.34; d 7, 24.05 ± 0.38; and d 14, 25.14 ± 0.45.

Article Snippet: Anti-m16 antibody and commercial NaPi2b antibody (positive control; sc-33928; goat polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA) were incubated on slides overnight at 1:100 dilution in normal antibody diluent (Fisher Scientific, Pittsburgh, PA).

Techniques: Sequencing, Control

Journal: The Journal of Biological Chemistry

Article Title: Convergent Signaling Pathways Regulate Parathyroid Hormone and Fibroblast Growth Factor-23 Action on NPT2A-mediated Phosphate Transport *

doi: 10.1074/jbc.M116.744052

Figure Lengend Snippet: NPT2A, NHERF1, PTHR, and FGFR1 expression in RPTECs. A, distribution of NPT2A (green), NHERF1 (red), and nuclei (blue) in filter-grown RPTECs. Single optical sections from the apical region of the cell are shown. Arrows, NPT2A association with cilia. B, magnified views of a single cell. C, x-z plane depicting PTHR (green) and nuclear (blue) staining of RPTECs grown on coverslips. D, FGFR1 (green) and F-actin (red) staining of RPTECs grown on filters. The x-z plane is shown.

Article Snippet: The cells were incubated overnight at 4 °C with mouse monoclonal anti-NHERF1 antibody (diluted 1:30 in block solution) and one of three primary anti-Npt2a antibodies (diluted 1:200 in block solution): rabbit anti-Npt2a antibody (a gift from Dr. Mark Knepper, National Institutes of Health) or goat anti-Npt2a (Santa Cruz, sc-33928).

Techniques: Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: Convergent Signaling Pathways Regulate Parathyroid Hormone and Fibroblast Growth Factor-23 Action on NPT2A-mediated Phosphate Transport *

doi: 10.1074/jbc.M116.744052

Figure Lengend Snippet: PTH and FGF23 down-regulation of NPT2A. A, NPT2A protein levels in RPTECs after a 2-h treatment with PTH or FGF23. A representative experiment is presented. B, immunofluorescence experiments depicting the change in localization of NPT2A in response to PTH or FGF23 treatment. RPTECs were left untreated (control) or treated for 2 h with 100 nm PTH or FGF23. Green, NPT2A; red, NHERF1; blue, nuclei. The x-z plane is depicted.

Article Snippet: The cells were incubated overnight at 4 °C with mouse monoclonal anti-NHERF1 antibody (diluted 1:30 in block solution) and one of three primary anti-Npt2a antibodies (diluted 1:200 in block solution): rabbit anti-Npt2a antibody (a gift from Dr. Mark Knepper, National Institutes of Health) or goat anti-Npt2a (Santa Cruz, sc-33928).

Techniques: Immunofluorescence