Smase

Primary cultures of hippocampal or cortical neurons were prepared from WT and ASM‐KO mouse embryos as described above. Where indicated AEA (Sigma, dissolved in ethanol) was added to the medium at 5–100 μM for 1 h, SM (Sigma, dissolved in ethanol) at 40 μM for 48 h, SMase (Sigma, dissolved in phosphate buffer saline) at 0.1 U/100 μl for 24 h, the CB₁ inhibitor SR141716 (Sigma, dissolved in DMSO) at 1 μM for 1 h, the NSM inhibitor GW4869 (Cayman Chemical, dissolved in DMSO) at 15 μM for 1 h, and the autophagy inhibitor bafilomycin (Enzo Life Sciences, dissolved in DMSO) at 0.1 μM for 24 h. In some instances, the following FAAH inhibitors were added for 1 h at a final concentration of 50 μM from stocks dissolved in DMSO: JNJ‐1661010 (Sigma), PF‐04557845 (MedChem), and URB597 (Selleckchem).
Primary skin fibroblasts were cultured in DMEM. PF‐04557845 (MedChem) was added to the medium for 1 h at final concentrations of 50 and 100 μM from stocks dissolved in DMSO.

Cells were washed with serum-free culture medium and incubated with different concentrations (1–10 mM) of methyl-β-cyclodextrine (MCD) (Sigma-Aldrich) or sphingomyelinase (SMase) (1–50 mUN) from Bacillus cereus (Sigma-Aldrich) for 1 hr at 37°C in a humidity-controlled incubator. After lipid raft disruption cells were washed and used in different procedures.

Ten units of lyophilized B. cereus sphingomyelinase (SMase, Sigma S7651) were dissolved in 200 μl of cold 50% glycerol in sterile Ca²⁺/Mg²⁺ free PBS, yielding 50 mU/μl. Because SMase loses activity even when stored at − 70⁰ C, it was best used fresh. Neurons were treated with vehicle control and SMase diluted to 2.5 mU/ml in Neurobasal/B27 for 2 h.