10 ml samples were centrifuged, washed with 10 ml RN media and 10 ml H2O. Pellets were resuspended in 100 μl H2O and lipids extracted through the addition of 360 μl of chloroform:methanol:HCl (1/2/0.02) and incubated at room temperature for 20 minutes. 120 μl chloroform and 120 μl 2 M KCl were added to separate
phases and after centrifugation, the organic phase was removed and radioactivity quantified by scintillation counting. Thin-layer chromatography Radiolabelled lipids were analyzed by 1-dimensional and 2-dimensional thin-layer chromatography. The 1-dimensional system used to separate phospholipids SBI-0206965 from diacylglycerol and fatty acid was Silica Gel G layers developed with chloroform:methanol:acetic acid (98/2/1) and visualized using Bioscan imaging detector. The 2-dimensional
system also employed Silica Gel G layers and was developed first with chloroform:methanol:water (65/25/4) and secondly tetrahydrofuran/dimethoxyethane/methanol/4 M ammonium hydroxide (10/6/4/1). The resulting thin-layer plate exposed Belnacasan ic50 to a PhosphoImager screen and visualized using a Typhoon 9200. Lipid mass spectrometry Mass spectrometry of phospholipids was performed using a Finnigan TSQ Quantum (Thermo Electron, San Jose, CA) triple quadrupole mass spectrometer. Samples were prepared in 50:50 (v/v) chloroform:methanol. The instrument was operated in the negative ion mode. Ion source oxyclozanide parameters were as follows: spray voltage
of 3,000 V, capillary temperature of 270°C, capillary offset of 35 V, and the tube lens offset was set by infusion of polytyrosine tuning and calibration solution (Thermo Electron, San Jose, CA) in the electrospray mode. Acquisition parameters were as follows: full scan, scan range 600 – 100 m/z, scan time 0.5 s, peak width Q1 0.7 FWHM. Instrument control and data acquisition was performed with the Finnigan™ Xcalibur™ software (Thermo Electron, San Jose, CA). Mass spectrometry malonyl-CoA measurement Cultures of strain PDJ28 were grown in RN medium supplemented with 0.1% glycerol to OD600 = 0.6. Cells were pelleted and washed with 50 ml RN medium to remove glycerol and used to inoculate RN medium with and without 0.1% glycerol. Cultures were grown for 120 minutes and harvested at room temperature. Cells were extracted using the Bligh and Dyer method , and 50 pmol of [13C3]malonyl-CoA (Stable Isotope Products; Isotec) was added. The aqueous phase was applied to a 100-mg 2-(2-pyridyl)ethyl functionalized silica gel column (Supelco) equilibrated with 2% acetic acid in methanol/water (1:1) . The column was washed two times with 1 ml of equilibration buffer and 1 ml water. CoAs were eluted with 1 ml of 50% acetonitrile containing 15 mM ammonium hydroxide. Mass spectrometry of acyl-CoA was performed using a Finnigan TSQ Quantum (Thermo Electron) 10058-F4 triple-quadrupole mass spectrometer .