Journal: Cell Death & Disease
Article Title: Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis
Figure Lengend Snippet: C 2 -ceramide induced activation and release of CTSB. ( a ) Cells were treated with 50 μ M C 2 -ceramide (C 2 -Cer) for 6 h. Ceramide amounts were measured by LC-MS/MS. Values are mean±S.D. from three different experiments. * P
Article Snippet: Materials Materials were purchased as follows: human recombinant IL-2 (Imunase 35) (Shionogi Pharmaceutical, Osaka, Japan); C6 -NBD ceramide, C6 -NBD sphingomyelin, and C2 -ceramide (Matreya, Pleasant Gap, PA, USA); CTSB specific inhibitor IV (CA-074 Me) (Merck Millipore, Darmstadt, Germany); pepstatin A and desipramine (Sigma-Aldrich, St. Louis, MO, USA); anti-caspase-8, anti-caspase-9, anti-PARP, antilysosomal-associated membrane protein 1 (Lamp1), anti-Lamp2, anti-ASM, anti-CTSB, anti-CTSD, and anti-β -actin antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); Annexin V-conjugated FITC and monoclonal antibody against XIAP (BD Biosciences, Beverly, MA, USA); antiactive caspase-3 (p17) antibody and horseradish peroxidase–conjugated secondary antibodies (Promega, Madison, WI, USA); anti-caspase-3, anti-pan-cadherin, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (Cell signaling, Danvers, MA, USA); and anti-ceramide monoclonal IgM Ab clone (NHCER-2) was produced previously in our lab.
Techniques: Activation Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry