Lewis AM , Masgrau R , Vasudevan SR , Yamasaki M , O'Neill JS , Garnham C , James K , Macdonald A , Ziegler M , Galione A , Churchill GC .

075203 Wellcome Trust , BB/D012694/1 Biotechnology and Biological Sciences Research Council

	Supplementary Figure 1.
	Supplementary Figure 2.
	Fig. 1. (A) Schematic diagram of the acid extraction process for NAADP. Sperm or eggs are harvested from L. pictus, or cells are prepared from male CD1 mouse pancreas. Agonist is incubated with the cell preparation, and then HClO4 is added to stop the reaction at the required time point. The sample is then sonicated to disrupt the cells and is centrifuged to pellet the protein for subsequent assay. The supernatant is neutralized with an equal volume of 2 M KHCO3, or other bases where indicated, in preparation for analysis using the radioreceptor binding assay. (B) Schematic diagram of the NAADP radioreceptor binding assay. First, known concentrations of NAADP (blue boxes), or cell extracts, are added, followed by sea urchin egg homogenate (pale blue shapes) in intracellular medium and a 10-min incubation period. NAADP binds irreversibly to the receptors in the homogenate. [32P]NAADP (red boxes) is then added. This binds to the remaining available receptor sites. Bound NAADP is separated from the mixture by filtration, and the radioactivity is determined. Sample NAADP concentrations may be determined from the standard curve. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 2. Stability of nucleotides during the acid extraction procedure. (A) Schematic showing the procedure. NAADP, NADP, or NADPH (1 mM) was incubated for 6 h in the solutions indicated. Samples were then neutralized by the addition of Hepes acid for basic samples, or Tris base for acidic samples, and were analyzed by HPLC. HPLC traces show the stability of NAADP, NADP, and NADPH in Hepes (pH 7.2) (B), HClO4 (pH 0.88) (C), HClO4 neutralized with KHCO3 (pH 8.6) (D), KHCO3 (pH 9.1) (E), and K2CO3 (pH 11) (F). HPLC traces are scaled to a common peak height for comparison (maximum 20% scaling). Note that NADP is stable except at pH 11, when a number of products result; in particular, NAADP is generated, and this would interfere with correct determination of NAADP levels from a cell extract. NADPH is stable only at pH 7.2, but NADP is the principal breakdown product; NAADP is not produced under the conditions tested. a.u., arbitrary unit.
	Fig. 3. Synthesis of [32P]NAADP. (A) HPLC traces of the test reactions using unlabeled compounds. First, NAD is converted to NADP using NAD kinase. Second, NADP is converted to NAADP using ADP-ribosyl cyclase. (B) Separation of the [32P]NAADP from the reaction mixture. Detection of unlabeled compounds is performed by UV detection at 254 nm. 32P-labeled compounds are detected as counts per minute (cpm) using an in-line Geiger counter.
	Fig. 4. Measurement of NAADP levels in pancreatic acinar cell samples. (A) Schematic diagram showing the preparation of pancreatic acinar cell samples. These samples were then diluted for use in the assay. (B) A competitive displacement curve was generated using standard concentrations of NAADP (black filled circles). Dilutions of the pancreatic acinar cell sample were determined from this curve and are shown on the trace (red triangles). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 5. Detection of changes in NAADP concentration under cell extract conditions. The displacement curve was generated from known NAADP concentrations (blue squares). The difference between known NAADP concentrations spiked into rat brain extract (prepared as described in text) is correctly detected (red circles). Error bars represent standard errors of the mean (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 6. Improvement of NAADP detection in cell extract samples. The green trace shows standards made in intracellular medium (Glu-IM), the blue trace shows standards in “acid-extracted” intracellular medium, and the red trace shows standards in intracellular medium supplemented with 1 M potassium acetate. Error bars represent standard errors of the mean (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 7. Detection of radioactivity using storage phosphor screens and a Typhoon scanner. (A) Comparison of radioactivity determined by the Typhoon scanner image versus counts per minute (cpm) detected using Cerenkov scintillation counting. (B) Image produced for a standard NAADP displacement curve using the Typhoon scanner . (C) Comparison of standard curves with radioactivity detected using scintillation counting (red symbols and line) and the Typhoon scanner (blue symbols and line). Error bars represent standard errors of the mean (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 8. BAPTA-AM, a cell-permeant form of the Ca2+ chelator BAPTA, prevents NAADP metabolism and, hence, facilitates NAADP measurements in pancreatic acinar cells. The red trace shows cells preincubated with 20 μM BAPTA-AM, followed by the addition of control buffer and incubations for the various times. The blue trace shows cells that were preincubated with BAPTA-AM, followed by addition of 100 pM CCK for the times indicated. Error bars represent standard errors of the mean (n = 3). The inset (green trace) shows an initial peak in NAADP levels in response to CCK in the absence of BAPTA. This response is very transient and, hence, difficult to detect. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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