Fig. 3.

CB₁R agonist-mediated redistribution of β-arrestin 1/2 is influenced by CRIP1a. A, Visualization of the redistribution of β-arrestin 1/2 by immunofluorescence microscopy. N18TG2 WT, empty vector control, CRIP1a XS, and CRIP1a KD cells were treated for 5 minutes with vehicle or CP55940 (10 nM); scale bar, 10 μm. (B) Quantification of β-arrestin 1/2 staining after 5-minute treatment with vehicle or CP55940 (10 nM). (B) Quantification of β-arrestin 1/2 staining after 5-minute treatment with vehicle or CP55940 (10 nM). Data are shown as average pixel intensity per cell after subtracting the background, from a total of 29 to 44 cells evaluated for each treatment group. The values were averaged for each treatment group and reported as the mean ± S.E.M. from n = three independent experiments. Statistical analysis was performed using 2-way ANOVA and Bonferroni multiple comparisons test. No significant differences in vehicle-treated samples were observed between WT, control, CRIP1a XS, and CRIP1a KD clones. *P < 0.05 indicates a significant difference between vehicle and CP55940-treatment. ##P < 0.01, ###P < 0.001 indicate significant difference from CP55940-treated WT. (C) Quantification of pixels in the high-intensity range as a fraction of total pixels. Using CellSens software, the total pixel number emitting fluorescence was determined after the sliding scale bar had been set to a minimum that was equivalent to the background. To determine the number of pixels emitting fluorescence in the high intensity range, we arbitrarily defined high intensity as “above the population of basal.” We calculated this nonbasal average intensity level to be one standard deviation above the mean of the average pixel intensity of all the vehicle-treated control cells (see gray area at the bottom of the bars in Fig. 3B). The sliding scale bar was moved to bring the minimum up to the nonbasal level, and the number of pixels emitting fluorescence in this high-intensity range was determined. The ratio of high intensity (i.e., above basal) pixels to total (i.e., above background) pixels was calculated as a percent.