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, 2010a; Graves et?al., 2008). Briefly, cell lysates were obtained by Dounce homogenization in a homogenizing buffer (HM buffer; 0.25?M sucrose, 1?mM EDTA, 10?mM HEPES; pH 7.0), and then centrifuged at 1,900?�� g (4, 200?rpm) PD0325901 concentration at 4��C for 10?min to remove the nuclei and intact cells. Postnuclear supernatants then underwent ultracentrifugation through a Percoll density gradient using a Beckman L8-70 ultracentrifuge. An ultracentrifuge tube was layered with 2.5?M sucrose, 18% Percoll in HM buffer. Centrifugation speed was 67,200?�� g (14,000?rpm) at 4��C for 1?hr using a Beckman Coulter 70.1 Ti rotor. Samples were fractionated into light, medium, and heavy membrane fractions. Heavy membrane fractions contained concentrated bands of cellular organelles and were further layered over a discontinuous iodixanol gradient. The iodixanol gradient was generated by mixing iodixanol in HM buffer with 2.5?M glucose (in?v/v; 27%, 22.5%, 19%, 16%, 12%, and 8%); the osmolarity of all solutions?was ?300 mOsm. After centrifugation at 4��C for 2.5?hr, the sample was divided into ten fractions (0.5?ml each) for biochemical and atomic absorption analyses. Note that the ionic composition of the lysosome was largely maintained due to the low rate of ion transport across DEF6 the lysosomal membrane at 4��C.?Antibodies used for western blots were anti-Lamp-1 (Iowa Hybridoma Bank), 1:5,000 dilution; anti-Annexin V (Abcam), 1:2,000 dilution; anti-GM130 (Abcam), 1:2,000 dilution; anti-EEA1 (Santa Cruz Biotechnology), 1:500 dilution; anti-Complex II (Abcam), 1:5,000 dilution; and anti-GFP (Covance), 1:5,000 dilution. Lysosomal fractions were prepared for atomic absorption by diluting the samples in a 1/1 ratio with concentrated nitric acid. After digestion (10?min, 60��C), the ionic composition was measured using a Thermo Scientific Finnigan Element inductively coupled with a plasma high-resolution mass spectrometer (S��by et?al., 2003). Data are presented as mean �� SEM. Statistical comparisons were made using ANOVA. A p value Selleckchem EPZ-6438 mutagenesis kit. All constructs were confirmed by sequencing, and protein expression was verified by Western blot and fluorescence imaging. COS-1 or HEK293T cells, used for all the heterologous expression experiments, were transfected using Lipofectamine 2000 (Invitrogen) with human TPC1, mouse TPC1, human TPC2, or mouse TPC2 fused with either EGFP or mCherry. Confocal images were taken using a Leica (TCS SP5) microscope and an Olympus Spinning-disk confocal system. Generation of TPC1 and TPC2 double knockout mice.