Protein bands detected on Western blots were quantified using ImageJ 1.47v software. Data were analyzed using GraphPad Prism 5 software. Densitometry of every protein band was carried out with ImageJ. We used the same area size to perform densitometry for every protein band from the same experiment for every phosphorylation site as well as the total receptor. Accordingly, an equally sized, empty area from the blot/film was measured to subtract this value as background signal from every measuring point. Last, phosphorylation signals were normalized to the total receptor (phosphorylation-independent antibody; NOP receptor). Controls (MOCK or SCR) were defined as 100%, and phosphorylation of every target protein was calculated as percentage phosphorylation in comparison to the respective control. Statistical analysis was carried out with one-way ANOVA followed by Bonferroni correction. P values <0.05 were considered statistically significant.

To compare the ability of different agonists to induce NOP-YFP internalization in primary cultures of ventral midbrain neurons, fluorescent puncta formation was used as a proxy for receptor internalization. This allowed analysis to be performed on images containing several neurons with many of overlapping neurites. It also allowed automated analysis over large numbers of images without the need to manually select regions corresponding to the plasma membrane and cell interior. Puncta identification and counting were performed using Python code that was based on the original approach and MATLAB code developed by Aguet et al. (126). To quantitatively compare the extent of puncta formation across different conditions, the number of puncta per image was normalized to the total NOP-YFP intensity. The plot shows this “puncta density” for each condition relative to N/OFQ.

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