Urb597

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.

The generation and initial characterization of PAK1 KO mice were described previously (Asrar et al., 2009 (link); Huang et al., 2011 (link)). All the mice used in this study were PAK1 KO (Pak1-/-) offspring and their wild type littermaze (Pak1+/+) derived PAK1 heterozygous (Pak1+/-) breeders to minimize the effect of genetic or environmental factors. The age of the animals in all experiments was 25–32 days. The following PCR primers were used for PAK1 mouse genotyping: (Pak1+F: 5’-CTGAGGGAAGAGACTGCAGAG-3’, Pak1+R: 5’-AGGCAGAGGTTTGGAGCCGTG-3’; Pak1-F: 5’-CTGAGGGAAGAGACTGCAGAG-3’, Pak1-R:5’-GGGGGAACTTCCTGACTAGG-3’). The absence of PAK1 in the PAK1 KO mice was confirmed by Western blot analysis. The mice were housed under a standard 12/12 light/dark cycle condition. All the procedures used for this study were approved by the Animal care committees at the Hospital for Sick Children, Canada and Southeast University, China. AM251, cytochalasin D (Cyto-D), JZL184, Nimesulide, NSC23766 (NSC) and URB597 were from Selleck; D-2-amino-5-phosphonovalerate (D-APV) and Glycyl-H 1152 dihydrochloride (GH1152) were from Tocris; all other drugs (NBQX disodium salt hydrate, (R)-(+)-WIN 55,212–2 mesylate salt [WIN], IPA3, TTX, and picrotoxin) were from Sigma.