Journal: The Journal of Biological Chemistry
Article Title: Kdo2-Lipid A, a TLR4-specific Agonist, Induces de Novo Sphingolipid Biosynthesis in RAW264.7 Macrophages, Which Is Essential for Induction of Autophagy *
Figure Lengend Snippet: KLA induces time-dependent increases in ceramide and dihydroceramide. RAW264.7 cells were incubated with vehicle control (PBS) or KLA (100 ng/ml). Following treatment, cells were harvested at the indicated time points for lipid extraction and analysis by LC ESI-MS/MS. A , amounts of the major chain length subspecies of DHCer. Data represent the means ( n = 9). B , amounts of total DHCer (a summation of all chain lengths) in KLA versus control conditions. Data represent the means ± S.E. ( n = 9); *, p ≤ 0.05; **, p ≤ 0.001. C , amounts of the major chain length subspecies of Cer. Because of the difficulties in visualization, the quantities of ceramide subspecies in control treated cells at the 24-h time point are as follows: C16 (9 pmol/μg DNA); C18 (0.4 pmol/μg DNA); C20 (0.2 pmol/μg DNA); C22 (1.9 pmol/μg DNA); C24:1 (5.4 pmol/μg DNA); C24 (5.5 pmol/μg DNA); C26:1 (0.2 pmol/μg DNA); and C26 (0.1 pmol/μg DNA). Data represent the means ± S.E. ( n = 9). D , amounts of total Cer (a summation of all chain lengths) in KLA versus control conditions. Data represent the means ± S.E. ( n = 9); *, p ≤ 0.05; **, p ≤ 0.001.
Article Snippet: Materials The suppliers for the reagents were as follows: Kdo2 -lipid A and the internal standard mixture for SL analysis by LC ESI-MS/MS (ceramide/sphingoid internal standard mixture II, LM-6005) (Avanti Polar Lipids, Alabaster, AL); [U-13 C]palmitic acid (98%) (Cambridge Isotopes, Andover, MA); fatty acid-free BSA (Calbiochem) ; Hoechst 33342 (Invitrogen); ISP1 (Biomol, Plymouth Meeting, PA); mouse monoclonal antibody to SPT1 (anti-LCB1), anti-BiP/GRP78, and anti-GM130 (BD Biosciences); rabbit monoclonal anti-HA tag and rabbit polyclonal LC3B antibody (Cell Signaling Technology, Boston); mouse monoclonal anti-Cer antibody clone 15B4 (Alexis Biochemicals, San Diego) This antibody clone has been successfully used in immunocytochemistry applications with no indication of nonspecific reactions with other lipids ( ) The secondary Alexa Fluor-conjugated F(ab)2 fragment goat anti-mouse antibody and goat anti-rabbit antibody were from Molecular Probes, Inc. (Eugene, OR.).
Techniques: Incubation, Mass Spectrometry