612 a Mannosidase‖l Fla TOPRO-3 S283A H285A H328A H332A S733A were incubated with anti-rabbit FITC 1: 1000(Vector Labs ). After three (v/v) protease inhibitor cocktail (Sigma). Cell debris were cleared washes, TOPRO-3(Invitrogen), diluted 1: 1000 in blocking buffer, was by centrifugation at 8200 g for Immunoprecipitation was added to cells to stain the nuclei. Slides were mounted in Vectashield performed by incubating lysate 30 Al of an anti-Flag M2 Vector Labs) and analyzed on a confocal microscope(Bio-Rad monoclonal antibody covalently bound to agarose beads(Sigma) Radiance 2000)using 488 nm, 543 nm, and 638 nm excitation and a for 2 h Immune complexes were washed three times with 50 mM 40x objective lens. Images were processed with Photoshop(Adobe). Tris(pH 7.5), 5% sucrose, 1 mM EDTA SMSI or SMS2 activity assay was then performed as previously described [2 Briefly, 50 mM 2.6. SMSI, SMS2-specific activity assay Tris-HCl(pH 7.4). 25 mM KCl, C6-NBD-ceramide(o1 ug/ul), and phosphatidylcholine(0.01 ug/y) was incubated with the immuno- Cells, transiently expressing Flag-tagged SMS1 or SMS2, were precipitate at 37 C for 45 min. Lipids were extracted in chloro- lysed in 200 mM NaCl, 50 mM Tris(pH 7.5). 1 mM EDTA, and 1% form: methanol (2: 1), dried under N2 gas, and separated by thinwere incubated with anti-rabbit FITC 1:1000 (Vector Labs). After three washes, TOPRO-3 (Invitrogen), diluted 1:1000 in blocking buffer, was added to cells to stain the nuclei. Slides were mounted in VectaShield (Vector Labs) and analyzed on a confocal microscope (Bio-Rad Radiance 2000) using 488 nm, 543 nm, and 638 nm excitation and a 40× objective lens. Images were processed with Photoshop (Adobe). 2.6. SMS1-, SMS2-specific activity assay Cells, transiently expressing Flag-tagged SMS1 or SMS2, were lysed in 200 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, and 1% (v/v) protease inhibitor cocktail (Sigma). Cell debris were cleared by centrifugation at 8200 g for 10 min. Immunoprecipitation was performed by incubating lysate with 30 μl of an anti-Flag M2 monoclonal antibody covalently bound to agarose beads (Sigma) for 2 h. Immune complexes were washed three times with 50 mM Tris (pH 7.5), 5% sucrose, 1 mM EDTA. SMS1 or SMS2 activity assay was then performed as previously described [2]. Briefly, 50 mM Tris–HCl (pH 7.4), 25 mM KCl, C6-NBD-ceramide (0.1 μg/μl), and phosphatidylcholine (0.01 μg/μl) was incubated with the immuno￾precipitate at 37 °C for 45 min. Lipids were extracted in chloro￾form: methanol (2:1), dried under N2 gas, and separated by thin Fig. 2 (continued). 612 C. Yeang et al. / Biochimica et Biophysica Acta 1781 (2008) 610–617