Journal: Journal of Biological Chemistry

Article Title: Involvement of Neutral Ceramidase in Ceramide Metabolism at the Plasma Membrane and in Extracellular Milieu

doi: 10.1074/jbc.m506827200

Figure Lengend Snippet: FIGURE 3. Effects of expression of wild-type and mutant neutral CDases on the generation of Sph and S1P in CHOP cells. A, schematic diagram of cDNA constructs used. Wild-type CDase (mouse neutral CDase tagged with Myc at the C terminus); SecCD (wild-type CDase having the secretory signal sequence of the mouse Vh chain instead of the signal/anchor sequence); ERCD (ER retention signal was added to the C terminus of the wild-type CDase). B, CDase activities and proteins expressed in cells and released in the medium. Cells were transfected with a plasmid vector containing wild-type or mutant CDase cDNAs. The cell lysates and the culture supernatant were subjected to an assay of neutral CDase activity using C12-NBD-Cer as a substrate, and the protein expression was determined by Western blotting using anti-Myc antibody. C, subcellular localization of wild-type and mutant CDases. Cells transfected with a plasmid vector containing wild-type cDNA or mutant CDase cDNA were fixed, permeablized with Triton X-100 () or not (), and stained with anti-Myc antibody followed by Alexa 598-labeled anti-mouse IgG (Molecular Probes). The Myc signal was examined under a fluorescent microscope. D, Sph and S1P contents in mutant CDase-overexpressing CHOP cells. Cells were transfected with a plasmid vector containing wild-type CDase or mutant CDase. Twenty-four hours after transfection, the medium was replaced with serum-free-conditioned medium from each transfectant, and then cells were treated with 100 milliunits/ml of SMase for 1 h. Cells were harvested and subjected to HPLC analysis for measurement of the amounts of Sph and S1P. Data were averaged from three independent experiments with S.D. Statistical analysis was done using Student’s t test, and the p values are indicated.

Article Snippet: After treatment with blocking buffer (5% skim milk in PBS) for 15 min, the sampleswere incubatedwith primary antibody at 4 °C for 1 day followed by secondary antibody at room temperature for 2 h. Immunostained samples were examined with a fluorescent microscope (Zeiss Axioskop 2 Plus microscope with an Axio-Cam CCD camera, Carl Zeiss) or a confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).

Techniques: Expressing, Mutagenesis, Construct, Sequencing, Transfection, Plasmid Preparation, Activity Assay, Western Blot, Staining, Labeling, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Involvement of Neutral Ceramidase in Ceramide Metabolism at the Plasma Membrane and in Extracellular Milieu

doi: 10.1074/jbc.m506827200

Figure Lengend Snippet: FIGURE 4. The site for generation of Sph and S1P mediated by neutral CDase in CHOP cells. A, CHOP cells were transfected with neutral CDase cDNA or empty vector. At 24 h after transfection, the medium was replaced with serum-free -MEM, and the cells were treated with 100 milliunits/ml of SMase for 30 min. Then, cells were washed twice with -MEM containing 1% or 0% BSA at 4 °C for 5 min, harvested, and subjected to HPLC analysis for measurement of the amounts of Sph and S1P. Data were averaged from three independent experiments with S.D. B, at 24 h after transfection with the CDase cDNA, the medium was replaced with serum-free -MEM containing 1% or 0% BSA, then cells were treated with 100 milliunits/ml of SMase for the periods indicated, and the culture supernatants were used for the determination of the amount of Sph by HPLC. Data were averaged from three independent experiments with S.D. C, immunocytochemical analysis of CHO cells (mock-transfected cells) and CHO-SPHK1 cells (CHO cells stably overexpressing SPHK1). Cells were fixed, permeabilized with Triton X-100 () or not (), and stained with anti-SPHK1 antibody followed by Alexa 488-labeled anti-rabbit IgG (Molecular Probes). The signal for SPHK1 was examined under a fluorescent microscope. D, the generation of Sph and S1P in CHO-SPHK1 and CHO cells after transfection with a CDase cDNA or an empty vector. Twenty-four hours after transfection, the medium was replaced with fresh serum-free -MEM, and then cells were treated with 100 milliunits/ml of SMase for 1 h. Cells were harvested and subjected to HPLC analysis for measurement of the amounts of Sph and S1P. Data were averaged from three independent experiments with S.D. Statistical analysis was done using Student’s t test, and the p values are indicated.

Article Snippet: After treatment with blocking buffer (5% skim milk in PBS) for 15 min, the sampleswere incubatedwith primary antibody at 4 °C for 1 day followed by secondary antibody at room temperature for 2 h. Immunostained samples were examined with a fluorescent microscope (Zeiss Axioskop 2 Plus microscope with an Axio-Cam CCD camera, Carl Zeiss) or a confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).

Techniques: Transfection, Plasmid Preparation, Stable Transfection, Staining, Labeling, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Involvement of Neutral Ceramidase in Ceramide Metabolism at the Plasma Membrane and in Extracellular Milieu

doi: 10.1074/jbc.m506827200

Figure Lengend Snippet: FIGURE 8. Internalization of S1P1 mediated by neutral CDase-dependent genera- tion of S1P. A, CHO cells stably expressing HA-tagged S1P1 (CHO-S1P1) were grown on glass coverslips, and then incubated for 5 h with serum-free medium. In parallel, SecCD or mock-conditioned medium containing 10% FBS was treated with 100 milliunits/ml of SMase at 37 °C for 5 h. CHO-S1P1 cells were then stimulated with SMase-treated condi- tioned medium as described above. The conditioned medium was diluted 2–100-fold with fresh medium before being used for stimulation. After 10 min of stimulation, cells were fixed, permeablized with Triton X-100, and stained with anti-HA polyclonal anti- body followed by Alexa 488-labeled anti-rabbit IgG. The HA signals for S1P1 were exam- ined under a fluorescent microscope. Arrowheads and arrows indicate S1P1 in the plasma membrane and internalized S1P1, respectively. B, to evaluate the S1P1 receptor internal- ization, more than 200 cells were selected randomly and the number of cells showing internalized receptors was counted under a fluorescent microscope. C, effects of DMS on the generation of S1P (panel a) and internalization of the receptor (panel b). SecCD- conditioned medium containing 10% FBS was treated with 100 milliunits/ml of SMase at 37 °C for 5 h with (final concentration, 20 M) or without DMS. Panel a, the amount of S1P determined by HPLC analysis. Panel b, receptor internalization in CHO-S1P1 cells stimu- lated with the 10-fold diluted conditioned medium for 10 min. The internalization of S1P1 was determined by the methods described in A and B. Data were averaged from three independent experiments with S.D.

Article Snippet: After treatment with blocking buffer (5% skim milk in PBS) for 15 min, the sampleswere incubatedwith primary antibody at 4 °C for 1 day followed by secondary antibody at room temperature for 2 h. Immunostained samples were examined with a fluorescent microscope (Zeiss Axioskop 2 Plus microscope with an Axio-Cam CCD camera, Carl Zeiss) or a confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).

Techniques: Stable Transfection, Expressing, Incubation, Staining, Labeling, Microscopy, Clinical Proteomics, Membrane, Concentration Assay