Bakouh N , Chérif-Zahar B , Hulin P , Prié D , Friedlander G , Edelman A , Planelles G .

	Figure 1. Phosphate-induced current in X. laevis oocytes.Phosphate-induced current (1 mM of Pi added in ND96) was measured at holding potential (Vc) of −50 mV in oocytes expressing NPT2a or CFTR alone or co-expressing NPT2a and CFTR; oocytes injected with water were used as control oocytes. A: Original tracings showing the current induced by the addition of 1 mM Pi (indicated by black bars) in the superfusing medium. The type of oocytes is indicated above the tracings. B: Summary of the results (means ± SEM) of the effect of 1 mM Pi in the different types of oocytes (indicated below the columns). Significance of the results *: P<0.05
	Figure 2. Kinetic analysis of inorganic phosphate transport in oocytes expressing NPT2a or co-expressing NPT2a and CFTR.Phosphate-induced currents (IPi) were measured (Vc =  −50 mV) in NPT2a-oocytes (empty circles) and in NPT2a+CFTR-oocytes (filled circles). The dashed lines through the points (means ± SEM from n  =  6 to 14, N  =  5) were calculated using SigmaPlot software (Systat software Inc., San Jose, CA). A: IPi was induced by increasing Pi concentration in ND 96, pH 7.5, as indicated in abscissa and normalized against extrapolated maximal current (IPimax). Data were fitted to Michaelis-Menten equation. Results, as means ± SD, were as follows: the apparent concentration of Pi substrate (Km) giving the half IPimax was not changed by CFTR expression (Km  =  0.07± 0.02 vs 0.06 ± 0.02 mM in NPT2a- and NPT2a+CFTR-oocytes, respectively), but IPimax was decreased by 40±3% (P <0.05). B: IPi was induced by 1 mM Pi at increasing Na+ concentrations (equimolary substituted by choline+, pH 7.5) and normalized against IPimax. Data were fitted to the modified Hill equation. Results, as means ± SD, were as follows: the apparent Km for Na+ substrate was 40.7 ± 14.6 in NPT2a-oocytes, not different from 41.8 ± 13.4 mM in NPT2a+CFTR-oocytes; IPimax was decreased by 39±7% in NPT2a+CFTR-oocytes compared to NPT2a-oocytes (P < 0.05). Inset: Effect of varying extracellular pH from 7.0 to 8.0 on the current induced by 1 mM Pi in NPT2a-oocytes (white column) and in NPT2a+CFTR-oocytes (black column). IPi was normalized against the Pi-induced current measured at pH 7.5 in NPT2a-oocytes from the same batch of oocytes. Results are shown as means± SEM, n  =  5 oocytes of each type.
	Figure 3. Immunodetection of Myc-NPT2a protein expressed in Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes.A: Myc-NPT2a expression in Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes (total or biotinylated proteins) was analyzed by Western blot using a polyclonal Myc antibody. H2O-injected oocytes were used as control. Actin expression was used as loading control of total proteins. The molecular weight of Myc-NPT2a is indicated. B: Left part: Quantification of the staining intensity of Myc-NPT2a in total lysate over the staining intensity of actin from Myc-NPT2a-oocytes (white column) and NPT2a+CFTR-oocytes (black column). Results are expressed as means ± SEM (N = 4). Right part: Quantification of the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a+CFTR oocytes (black column) normalized against the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a-oocytes. Results are expressed as means ± SEM (N = 4); *: P<0.05.
	Figure 4. Effect of a permeant cAMP analog on the phosphate-induced current in NPT2a-oocytes or NPT2a+CFTR-oocytes.Phosphate-induced current (IPi) was measured in voltage-clamped (−50 mV) NPT2a- or co-and NPT2a+CFTR-oocytes. A. Original tracings obtained from a NPT2a-oocyte (left), and from a NPT2a+CFTR-oocyte (right), exposed in a reversible manner to 1 mM Pi, as indicated by a black bar. As indicated below the tracings, IPi was first induced in basal condition, basalIPi (superfusion of ND96 supplemented with 1 mM Pi), then in an experimental condition, exptlIPi, herein after 10 min exposure to 8 Bromoadenosine-3′5′-cyclic monophosphate (8-Br-cAMP, 100 µM). The break in each trace represents a ∼ 9 min exposure to the cAMP agonist that is not shown. B. Summary of the results (means ± SEM) of the effect of 8-Br-cAMP, 100 µM, on IPi in NPT2a-oocytes (n  =  8, white columns) and in NPT2a+CFTR-oocytes (n =  8, black columns) from N =  3 experiments. Measured IPi were normalized against basalIPi from NPT2a-oocytes from the same batch of oocytes. The significance of the difference between Pi-induced currents measured in basal or in experimental conditions in NPT2a+CFTR-oocytes and basalIPi from NPT2a-oocytes was analyzed using unpaired Student’s t-test (*: P < 0.05). The significance of the difference between basalIPi and exptlIPi within the same type of oocytes was assessed using paired Student’s t-test (#: P < 0.05).
	Figure 5. Effects of agonists of EPAC and PKA pathways on NPT2a function and surface expression.Phosphate-induced currents (IPi) were evoked in voltage-clamped (–50 mV) cells by the exposure of NPT2a-oocytes (white columns), or NPT2a+CFTR-oocytes (black columns) to 1 mM Pi. As above, IPi was first induced in basal condition, basalIPi, then after the activation of a cAMP-dependent signaling pathway, exptlIPi, during 10 min (panels A and B). Results are as means ± SEM. IPi were normalized against basalIPi from NPT2a-oocytes from the same batch of oocytes. The significance of the difference between basalIPi from NPT2a-oocytes and basalIPi or exptlIPi from NPT2a+CFTR-oocytes was analyzed using unpaired Student’s t-test (*: P < 0.05). The significance of the difference between basalIPi and exptlIPi within the same type of oocytes was assessed using paired Student’s t-test (#: P < 0.05). A: Effect of para-Chlorophenylthio-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP, 25 µM), an activator on the EPAC pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n  =  9 for each type; N =  2). B: Effect of N6-monobutyryladenosine-3′, 5′-cyclic monophosphate (6-MB-cAMP, 25 µM), an activator of the PKA pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n  =  19 and n  =  22, respectively; N =  6).
	Figure 6. Effect of PKA stimulation on NPT2a and CFTR cell surface expression.Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes from a same batch were kept in control condition or submitted to 25 µM of 6-MB-cAMP for 15 min, as indicated below the panel. Control oocytes were H2O-injected. A: Effect of PKA stimulation on NPT2a cell surface expression. Left: Cell surface biotinylated proteins were probed with an anti-Myc antibody; the molecular weight of Myc-NPT2a is indicated on the figure. Right: Results from 4 separate experiments, were quantified. The staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a+CFTR oocytes (black columns) was normalized against the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a-oocytes (white columns) in basal condition. The significance of the difference between the results was assessed using unpaired (*: P < 0.05) or paired (#: P < 0.05) Student’s t-test. B: Effect of PKA stimulation on CFTR cell surface expression. Cell surface biotinylated proteins were probed with an anti-CFTR antibody; the molecular weight of CFTR is indicated on the figure. Similar results were obtained in 2 independent experiments.
	Figure 7. NPT2a and CFTR expressed in X. laevis oocytes co-immunoprecipitate.Representative experiment showing the co-immunoprecipitation of NPT2a and CFTR after their expression in X. laevis oocytes. Proteins from oocytes co-expressing Myc-NPT2a and CFTR were immunoprecipitated with MM13-4 Ab, or with IgG1 Ab used as a negative control. They were probed with the anti-Myc antibody. The molecular weight of the detected protein is indicated Similar results were obtained in N  =  3 independent experiments.
	Figure 8. Effect of injecting NEG2 peptide on NPT2a function.The current induced by 1 mM phosphate (IPi) was measured in voltage-clamped (−50 mV) NPT2a-oocytes and was measured in oocytes injected either with the NEG2 peptide (black column, n  =  12), with a scrambled peptide (sNEG2, grey column, n  =  9), or in non-injected oocytes (white column, n  = 10). IPi was measured in basal condition (basalIPi, measured in ND96 medium) then under an experimental stimulating condition (exptlIPi, measured after a 10 min-incubation in ND96 supplemented with forskolin, 1µM, and isobutylmethylxanthine, 100 µM, Forsk+IBMX). Results are presented as means ± SEM from N  =  4 experiments. IPi were normalized against basalIPi from control (non injected with a peptide) NPT2a-oocytes from the same batch of oocytes. The significance of the difference was analyzed using unpaired Student’s t-test (*: P < 0.05) or paired Student’s t-test (#: P < 0.05).

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