Figure 2.

Subsynaptic compartmentalization of the CB1 receptor, the canonical transducers coupled to CB1 receptors and other proteins of the endocannabinoid system in PAZ, PSD, and EXTRA fractions isolated from cortical synaptosomes derived from wild-type mice. Representative Western blots carried out by immunoblotting increasing amounts of cortical synaptosomes (3, 6, 9, and 12 µg/lane) and different subsynaptic fractions of wild-type mice (3 µg/lane) using antibodies against CB1 receptor, Gαi/o subtypes, Gβ and Crip1a (A) and Gαq/11, PLC-β1, DAGL-α, and MAGL (B). Presynaptic fraction PAZ, postsynaptic fraction PSD, and extrasynaptic fraction EXTRA. Protein migration was consistent with their expected molecular mass. For the CB1 receptor and the Gαi2 protein, extra bands migrating at ~35 kDa and ~36 kDa were detected, respectively (CB1, 52.8 kDa; Gαo 40.1 kDa; Gαi1, 40.5 kDa; Gαi2, 40.4 kDa; Gαi3, 40.5 kDa; Gβ (common), 37.3 kDa and 36.3 kDa 1 and 2 isoforms; CRIP1a, 18.6 kDa; Gαq/11, 42.0 kDa; PLC-β1, 138.3 kDa and 133.3 kDa, the β1a and β1b isoforms, respectively; DAGL-α, 115.3 kDa; MAGL, 33.3 kDa). The molecular weights depicted correspond to the signal of the standard markers. (C) The bar graphs show the subsynaptic distribution of the CB1 receptor immunoreactive signals of ~50 kDa and ~35 kDa bands obtained with the CB1-Immunogenes, CB1-Go-Af450 and CB1-Rb-Af380 antibodies. The quantification was performed using data from the three antibodies together. The immunoreactive signals of PSD and EXTRA fractions are shown normalized to the total signal detected in both compartments. ~50 kDa: EXTRA 67.9 ± 0.7 vs. PSD 32.0 ± 0.7; ~35 kDa: EXTRA 52.8 ± 3.9 vs. PSD 47.9 ± 4.8. Values correspond to the means ± SEM of five independent assays, using subsynaptic fraction preparations obtained from a pool of cerebral cortices of eight adult mice. Unpaired two tailed t test. *** = p < 0.001.