Figure 2.

ERK 1/2 activation by CMV or TK promoter-driven full-length and Δ64-CB₁ receptor variants. (A) Western blot analysis of ERK 1/2 phosphorylation mediated by the various CMV and TK promoter-driven or endogenous CB₁ receptors stimulated with ACEA. Neuro 2a, HEK 293 and GT1-7 cells expressing the indicated CB₁R variants or endogenous receptors (Neuro 2a) were stimulated with various concentrations of ACEA in at 37°C in DMEM + HEPES for 5 min. Representative western blots are shown; please note that, although presented as separate blots, p-ERK and t-ERK membranes were actually developed under identical conditions (incl. exposure times) so these images may be directly compared within the pertinent cell type. (B) Dose-response analysis of ACEA-evoked p-ERK 1/2 signals from western blot experiments as the one presented on Panel (A). Phospho- to total ERK 1/2 values were normalized to the minimum response of the TK-CB₁R group and the 3-parametered log[agonist] – response equation was used to fit concentration-response curves. Number of observations was 3-8/construct/ACEA concentration; *p = 0.036 for the effect of CMV promoter on basal ERK activity and p = 0.19 for the effect of CMV promoter on EC₅₀ when compared to TK promoter in Neuro 2a cells; ^#p = 0.0002 for the effect of CMV promoter on basal phosphorylation and ^$p = 0.0288 for the effect of CMV promoter on EC₅₀ value vs. TK promoter in HEK 293 cells (2-way ANOVA).