Journal: Journal of Biological Chemistry

Article Title: O-Glycosylation of Mucin-like Domain Retains the Neutral Ceramidase on the Plasma Membranes as a Type II Integral Membrane Protein

doi: 10.1074/jbc.m207932200

Figure Lengend Snippet: FIG. 2. Immunocytochemical analysis of the neutral CDase overexpressed in HEK293 cells. A, HEK293 cells overexpressing GFP-fused neutral CDase. Cells were fixed and examined for GFP fluorescence under a confocal laser microscope. Arrows and an arrow- head indicate the expression of CDase at plasma membrane and ER/ Golgi compartments, respectively. B, HEK293 cells expressing GFP- fused neutral CDase (left) were fixed, and immunostained with anti- Rab6 antibody followed by anti-rabbit IgG-Cy3 (center). Images were merged (right). An arrow indicates the co-localization of neutral CDase and Rab6, a marker protein for the Golgi apparatus. C, analysis of membrane topology of neutral CDase. a, stained with anti-FLAG anti- body after permeabilization with Triton X-100. b, same as a but before treatment with Triton X-100. c, stained with anti-myc antibody after permeabilization with Triton X-100. d, same as c but before treatment with Triton X-100. HEK293 cells were transformed with plasmid vector containing FLAG-tagged CDase cDNA, fixed, and then stained with corresponding antibody before and after treatment with Triton X-100 as described under “Experimental Procedures.”

Article Snippet: After treatment with blocking buffer (5% skim milk in PBS) for 15 min, the samples were incubated with primary antibody (diluted 1:1000 with blocking buffer) at 4 °C for 1 day followed by Cy3-labeled secondary antibody at room temperature for 2 h. Immunostained samples were examined with a confocal laser-scanning microscope (Digital Eclipse C1, Nikon, Japan).

Techniques: Fluorescence, Microscopy, Expressing, Clinical Proteomics, Membrane, Marker, Staining, Transformation Assay, Plasmid Preparation