Fig. 1

Inhibition of ABHD6 by WWL70 increased the 2-AG levels in BV2 cells. a BV2 cells were treated with WWL70 or MAFP for 30 min and then incubated with ³[H]-2-AG at 37 °C for 2 min. Cell lysates were mixed with MeOH/CHCl₃ to separate the organic from aqueous phase. The amount of ³[H]-glycerol, the product of 2-AG hydrolysis, was measured in the aqueous phase. Inhibitors were added at either 1 or 10 μM. Data are represented as mean ± SD (n = 3). Triple asterisks denote p < 0.001 compared to the drug-treated groups. b The membrane fraction from BV2 cells was incubated with WWL70 for 10 min, reacted with HT-01 probe (1 μM) at 37 °C for 40 min, and then applied to SDS-PAGE. The gel was scanned to detect active ABHD6, followed by western blotting with an anti-calnexin antibody. c BV2 cells were incubated with 10 μM of WWL70 or MAFP for 1 h before harvest. Cell lipids were extracted with acetonitrile plus 0.02% TFA together with 2-AG-d₈ as an internal standard. 2-AG was identified and quantified with LC-MS/MS based on the ratio to the internal standard. Data are represented as mean ± SD (n = 6). Double and triple asterisks denote p < 0.01 and 0.001, respectively, compared to control