Primers for qPCR.

<p>1. Published by Nijenhuis <i>et al.</i>, 2003 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028166#pone.0028166-Nijenhuis1" target="_blank">[18]</a>.</p><p>2. On Demand TaqMan™ Gene Expression Assays (Applied Biosystems, Life Technologies Corporation).</p

TRPV6 mRNA expression in human and murine cells and tissues.

<p>A: Variation in reference gene expression in human cDNA templates. B: TRPV6 mRNA expression in human cells, relative to B2M. D3: 12 h with 10 nM 1,25D3. Data are individual measures with mean ± SEM. C: TRPV6 mRNA expression in murine duodenum and in primary murine calvarial osteoblasts, relative to B2M (n = 3 for duodenum and n = 4 for calvarial osteoblasts). * <i>P</i><0.05, n.s. not statistically significant.</p

TRPV6 protein expression in bone cells and tissues.

<p>A, B: TRPV6 expression in LNCaP (A) and TE-85 (B) cells. An inset image shows staining with normal goat IgG (1 µg/mL) in each cell line. The slides in A and B were processed simultaneously. C: Staining with normal goat IgG (1 µg/mL) in mouse bone. D, E: TRPV6 expression in normal murine duodenum (positive control). F-I: TRPV6 expression in normal murine bone. Tissues from 6-week old male C57Bl/6J mice were fixed in 10% buffered formalin for 48 hours. Bones were decalcified for 4 weeks in EDTA/PFA. Images are representative of three mice. Slides in C-I were processed simultaneously. Scale bar: 50 µm in all panels.</p

Currents in whole cell voltage-clamped SaOS-2 and 16HBE14o- cells.

<p>A, B: typical traces obtained from the same SaOS-2 cell in 2 mM [Ca²⁺]_O without (A) and with (B) 100 µM ruthenium red. C: Currents for SaOS-2 cells (mean ± SEM; n = 5) in 2 mM [Ca²⁺]_O without (open symbols) and with (solid symbols) 100 µM ruthenium red. A straight line adequately fits both conditions (<i>P</i> = 0.21; comparison of both datasets using the extra sum of squares F-test). D: Currents for 16HBE14o- cells (mean ± SEM; n = 15) in 2 mM [Ca²⁺]_O without (open symbols) and with (solid symbols) 100 µM ruthenium red. (<i>P</i> = 0.027; extra sum of squares F-test).</p

Effect of disruption of the NDPK-B/CFTR complex on the magnitude of FSK-dependent IDIDS and ICFTR in 16HBE14o- cells and the ACh-induced SCC in intestinal biopsy.

<p><b>a)</b> Effect of peptide NDPK-B 36–54 on ICFTR and IDIDS. Cells were incubated with NDPK-B 36–54 (100 μM) for 30 min prior to exposure to FSK/IBMX plus peptide NDPK-B 36–54 for 30 min. Control currents for each dataset were time and day matched. <b>b)</b> Lack of effect of peptide NDPK-A 36–54 on the outward DIDS-sensitive and CFTR_ihn172- sensitive conductances. Cells were incubated for 30 min in the presence of the peptide (100 μM), followed by incubation with the peptide plus FSK and IBMX for 30 min. Control currents for each dataset were time and day matched. <b>c</b>) Effect of peptide NDPK-A or NDPK-B 36–54 on SCC in gut epithelia. SCC measurements obtained in response to ACh (Cl^- flux) and glucose (Na⁺ flux) stimulation of mounted gut epithelia biopsies. Measurements were taken in the presence or absence of the synthetic peptides NDPK-A or NDPK-B 36–54 (100 μM) (n = 3) **P<0.05 ANOVA.</p

PKA regulates translocation of NDPK-B in airway epithelial cells.

<p><b>a)</b> Distribution of NDPK A and NDPK B in 16HBE14o- cells. Western Blot of membrane and cytosol (50 μg) from 16HBE14o- cells separated on a 15% SDS PAGE gel and transferred to PVDF membrane (Left panel, probed with NDPK B rabbit polyclonal antibody (1/5000); Middle panel, probed with NDPK B monoclonal antibody (1/1000); Right panel, probed for NDPK A). <b>b)</b> NDPK B is not detected in AMPKα immunoprecipitates. Left panel, western blot of AMPKα (pan) probed for NDPK A. Right panel, immunoprecipitates of AMPKα1 and AMPKα2 probed for NDPK B. <b>c)</b> Absence of complex between NDPK A and B in airway epithelia. Left panel, immunoprecipitates of NDPK B and NDPK A probed for NDPK A. Right panel, immunoprecipitates of NDPK A and NDPK B probed for NDPK B. Antibody staining detected with HRP antibody and a chemiluminescent substrate. Results representative of at least four independent experiments. Modulation of PKA activity alters localisation and distribution of NDPK-B in HNE. Immunocytochemical staining of HNE for NDPK-B in cells: <b>d)</b> untreated, <b>e)</b> treated with FSK/IBMX for 30 min, <b>f)</b> treated with PKI (5 min) prior to FSK/IBMX for 30 min, <b>g)</b> No primary antibody control. Bar is 5 μM and arrows show position of apical membrane. The result is representative of three independent experiments.</p

NDPK-B binds cell surface CFTR in forskolin/IBMX stimulated cells.

<p><b>a</b>) Immunoblots of avidin-agarose precipitates from lysates of 16HBE14o- cells ± FSK or PKI/ FSK/IBMX, biotinylated for 30 min at 4°C and probed for CFTR, NDPK-A and NDPK-B. <b>b</b>) Non-cell surface CFTR does not associate with NDPK-B. Immunoblots of CFTR immunoprecipitates from lysates of 16HBE14o- cells ± FSK or PKI/FSK/IBMX (post the avidin precipitation described in A, above) probed for CFTR and NDPK-B. <b>c</b>) NDPK-B does not associate with non-cell surface CFTR. Immunoblots of NDPK-B immunoprecipitates from lysates of 16HBE14o- cells ± FSK or PKI/FSK/IBMX (post the avidin-agarose precipitation described in A, above) probed for CFTR and NDPK-B. To confirm equal loading of the immunoprecipitates, blots were stripped and re-probed with CFTR rabbit polyclonal antibody (R & D systems) (B) or NDPK polyclonal antibody (C). The results are representative of two independent experiments.</p

Analysis of NDPK-B interaction with CFTR.

<p><b>a)</b> Position of the exposed side-chains of peptide 36–54, based on the published crystal structure of the NDPK-B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149097#pone.0149097.ref081" target="_blank">81</a>]. The CPK code was used: nitrogen is blue and oxygen is red. One subunit is shown in cyan to identify its border. Figure generated with RASMOL. <b>b)</b> Immunoblots of CFTR immunoprecipitates from lysates of 16HBE14o- cells treated with FSK/IBMX, and probed for NDPK-B and CFTR show peptide NDPK-B 36–54 (lanes 3, 4), but not peptide NDPK-A 36–54 (lanes 5, 6), released NDPK-B from CFTR immunoprecipitate. Control incubations with buffer alone are shown in lanes 1, 2. <b>c)</b> Peptide NDPK-B 36–54 releases CFTR from complex with NDPK-B. Immunoblots of NDPK-B immunoprecipitates from lysates of 16HBE14o- cells treated with FSK/IBMX, and probed for CFTR and NDPK-B show peptide NDPK-B 36–54 (lanes 3, 4), but not buffer alone control (lanes 1, 2) or peptide NDPK-A 36–54 (lanes 5, 6), released CFTR from NDPK-B immunoprecipitate.</p

NDPK-B /NBD1 interaction analysis of by Surface Plasmon Resonance.

<p><b>a</b>) Membrane (PVDF) was spotted with purified NDPK-B (lane 1), GST-NBD1 (351–727), R domain (635–837) and GST-NBD2 (1151–1360) was overlaid with purified recombinant NDPK-B and probed for NDPK-B. Purified NDPK-B bound to GST-NBD1. <b>b</b>) Coomassie blue staining of PVDF membrane showing the purity of NBD1 (28 kDa) (lanes 1–3; 900, 300 and 100 ng per lane, respectively) and NDPK-B (lane 4, 16 kDa) and the amounts of the proteins loaded. <b>c</b>) Overlay analysis of the direct interaction between the NBD1 domain (351–727) of CFTR and NDPK-B. Overlay experiment showing western blot containing NBD1 (lanes 1–3; 900, 300 and 100 ng per lane, respectively), NDPK-B (250 ng, lane 4) and BSA (250 ng, lane 5) overlaid with purified NDPK-B (50 μg) and then probed for NDPK-B staining using NDPK-B specific antibodies. BSA was used as a control to exclude non-specific interactions with NDPK-B. <b>d)</b> SPR analysis showing the direct interaction between the NBD1 of CFTR and NDPK-B. Example of sensorgrams obtained when several quantities of His-tagged NBD1 (10, 20, 40 or 60 ng) were injected over immobilized NDPK-B proteins. In the inset, the RU = f(ng) curve shows that the RU values obtained twenty seconds into the dissociation phase linearly increase with the amount of injected NBD1. <b>e)</b> SPR analysis showing the repeatability of the measurement of the direct interaction between the NBD1 of CFTR and NDPK-B. Example of sensorgrams obtained from two separate measurements when 40 ng of His-tagged NBD1 were injected over immobilized NDPK-B proteins. <b>f)</b> SPR analysis showing the specificity of the interaction between the NBD1 of CFTR and NDPK-B. Example of sensorgrams obtained when 1 μg of untagged NBD1 was injected alone or with several quantities of NDPK-B peptide (10, 50 or 100 ng) over immobilized NDPK-B proteins. A sensorgram obtained when 200 ng of NDPK-B peptide was injected alone over immobilized NDPK-B proteins is also shown. <b>g)</b> SPR analysis showing the NDPK-B key residues for the NBD1-NDPK-B interaction. Example of sensorgrams obtained when 1 μg of untagged NBD1 was injected alone or with 100 ng of different mutated NDPK-B peptides over immobilized NDPK-B proteins. Sensorgrams obtained when 1 μg of untagged NBD1 was injected with 100 ng of NDPK-A or NDPK-B peptide over immobilized NDPK-B proteins are also shown.</p

Forskolin (FSK) enhances NDPK-B interaction with CFTR in 16HBE14o- cells

<p><b>a)</b> PKA regulates co-immunoprecipitation of CFTR with NDPK-B in 16HBE14o-. Immunoblot of CFTR immunoprecipitate from 16HBE14o- membranes showing that FSK increases the amount of NDPK-B, but not NDPK-A, which co-immunoprecipitates with CFTR. Cells were untreated (lane 1), treated with FSK/IBMX for 30 min (lane 2) or with PKI for 5 min prior to FSK/IBMX treatment (lanes 3) and probed for NDPK-A and NDPK-B. Equal loading of the CFTR immunoprecipitate, was confirmed by re-probing the same blot for CFTR using a polyclonal antibody (R & D systems). <b>b)</b> Quantification of the band density of NDPK-B and NDPK-A shows a 3-fold increase in NDPK-B co-immunoprecipitation with CFTR with FSK/IBMX (n = 4). * P<0.05 Student <i>t</i>-test. <b>c)</b> Immunoblot of NDPK-B immunoprecipitate from 16HBE14o- membrane from cells: untreated (lane 1); treated with FSK/IBMX (lane 2) or PKI (5 min) prior to FSK/IBMX for a further 30 min (lane 3) and probed for CFTR. Equal loading of immunoprecipitate was confirmed by re-probing the same blot for NDPK-B polyclonal antibody. PKI inhibited the impact of FSK/IBMX on NDPK-B co-immunoprecipitation with CFTR. <b>d)</b> Quantitative analysis of the CFTR band density shows a 2-fold increase in CFTR co-immunoprecipitation with NDPK-B with FSK/IBMX (n = 4) * P<0.05 Student <i>t</i>-test.</p