Figure 4. Peroxide-dependent inhibition of MGL activity and 2-AG accumulation in brain neurons.

(A-D) Effects of H₂O₂ (filled bars) or vehicle (open bars) on (A) MGL activity, (B) 2-AG levels, and (C) 1-stearoyl,2-arachidonoylglycerol (SAG), arachidonic acid (AA), and anandamide (AEA) in rat cortical neurons in primary cultures. (D) Detection of sulfenylated MGL (MGL_Sfe) in primary cortical neurons in cultures after H₂O₂ treatment (300 μM, 1h). Sulfenylated MGL was trapped using the chemoselective probe BP1. Equal sample loading to the affinity precipitation was confirmed by measuring MGL (anti-MGL antibody) and actin (input controls). 50, 25: molecular weight size markers (kDa). (E) Ion current of the BP1-adduct of MGL peptide bearing C201 (590.53 m/z, z=4) extracted from the control incubation (black trace) and incubation of neurons with H₂O₂ (red trace). (F) Ion current of the BP1-adduct of MGL peptide bearing C208 (606.30 m/z, z=3) extracted from the control incubation (black trace) and incubation of neurons with H₂O₂ (red trace). In both E and F, the high-resolution mass spectra reported in the inset matches the expected charge state and m/z value. ***P<0.001 and *P<0.05 compared to vehicle, two-tailed Student’s t test.