Fig 2: Panoramic micrographs of intact nuclei isolated from the adult rat brain cortex processed for MGL-, ABHD12- and COX2-immunofluorescence (A–C) combined with NeuN/Fox-3-immunofluorescence (D–F) and Hoechst’s chromatin staining (G–I). Every NeuN-positive nucleus exhibited strong immunoreactivity for MGL, ABHD12 and COX2 (filled arrowheads), whereas NeuN-negative nuclei (empty arrowheads) displayed clear positive staining only for COX2. All micrographs were acquired in grayscale and pseudo colored. Scale bar = 20 µm in I (applies to (A–I)).

Fig 3: Analysis of the subcellular expression of 2-AG metabolizing enzymes monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH), a/ß hydrolase domain-containing protein 12 (ABHD12) and cyclooxygenase-2 (COX2). (A) Representative immunoblots of whole homogenate (WH) intact nuclear (N), plasma membrane (P2), microsomal (P3) and cytosolic (S3) fractions obtained from homogenates of the adult rat brain cortex. 20 µg protein from each fraction were loaded in the same gel. Incremental amounts of P2 membranes ranging from 5 to 40 µg protein were also run in parallel to generate reference standard curves used for semi-quantitative densitometric analysis of protein expression. (B) Graphs on the left correspond to standard curves of MGL-, FAAH-, ABHD12- and COX2-immunoreactivity in P2 samples. All optical density (OD) values were normalized to the value obtained at the highest protein loading. The correlation coefficients obtained by linear regression analysis are shown. Bar graphs on the right show results of the semi-quantitative analysis of MGL, FAAH, ABHD12 and COX2 protein expression in N, P2, P3 and S3 fractions. Values correspond to the percentage of the expression found in WH (100%, dashed line). Data presented are means ± SEM of four independent experiments (n = 4), except for FAAH (n = 2).

Fig 4: High-power micrographs of intact nuclei isolated from the adult rat brain cortex double stained with anti-ABHD12 (green) combined with Hoechst staining (blue) and NPCx ((A–E); red), NeuN/Fox-3 ((F–J); red) and DGLa ((K–O); red) immunostaining. All micrographs are maximum intensity projections of three consecutive 0.24 µm-thick optical sections acquired in grayscale and pseudo colored. Scale bar = 5 µm in O (applies to (A–O)).

Fig 5: Characterization of nuclear subfractions obtained from intact nuclei and analysis of nuclear compartmentation of MGL, ABHD12, COX2. (A) Bar graph depicting the partitioning of total protein from cortical intact nuclei (N) into nucleoids (Nu), nuclear matrix (NM), TX-100 extractable supernatant (S1) and DNase I/high salt extractable supernatant (S2). Data are expressed as percent protein recovered in each fraction relative to total protein (100%) in N fraction. Values are mean ± SD of two independent experiments. (B) Coomassie blue-stained SDS-PAGE of equivalent amounts (20 µg) of protein from N, Nu, NM, S1 and S2 samples. (C) Western blot analysis of nuclear subfractions (12 µg/lane) run in parallel to analyze the suitability of the subfractioning procedure using specific markers for non-ionic detergent extractable (BiP), non-ionic detergent and DNase I/high salt resistant (NPCx), nuclear matrix (NeuN/Fox-3) and DNA-bound (Histone H3) proteins. (D) MGL, ABHD12 and COX2 immunoblots in N, Nu, NM, S1 and S2 nuclear subfractions. 12 µg/lane were loaded.