Ins(1,3,4,5)P4 was prepared according to41 . Ins(1,3,4)P3 and Ins(1,4,5)P3 were obtained from AG Scientific as K+ salts. Ins(3,4,5)P3 was obtained from SiChem as Na+ salt. All biphenyl phosphates and related compounds were prepared as described23 (link), 24 (link) or in a very similar fashion. AS 1949490 (7 ) was obtained from Tocris. Sodium pyrophosphate, geranyl pyrophosphate and isopentenyl pyrophosphate were obtained from Sigma-Aldrich. Di-C8-PtdIns(3,4,5)P3 was obtained from Echelon Biosciences.
All compounds biologically evaluated had > 95% purity as determined by reverse-phase hplc on a 4.6 x 250mm Phenomenex Synergi Hydro-RP column eluted isocratically at 1 ml.min-1 with a solvent mixture comprising 4mM tetrabutylammonium hydroxide in 70/30, v/v, 50 mM NaH2PO4/ (acetonitrile/MeOH/water, 40/50/10, v/v/v). Samples, 10 μL of 10 μM compound were injected and peaks were detected with a Jasco FP-950 fluorescence detector set at Ex 285nm, Em 320nm and gain 10.
All compounds biologically evaluated had > 95% purity as determined by reverse-phase hplc on a 4.6 x 250mm Phenomenex Synergi Hydro-RP column eluted isocratically at 1 ml.min-1 with a solvent mixture comprising 4mM tetrabutylammonium hydroxide in 70/30, v/v, 50 mM NaH2PO4/ (acetonitrile/MeOH/water, 40/50/10, v/v/v). Samples, 10 μL of 10 μM compound were injected and peaks were detected with a Jasco FP-950 fluorescence detector set at Ex 285nm, Em 320nm and gain 10.
