Morphine desensitization, internalization and down-regulation of the mu opioid receptor is facilitated by serotonin 5-HT2A receptor co-activation

Data Supplement

Files in this Data Supplement:

• Data Supplement - Supplementary Figure 1. Controls for specificity of the hybridization signal obtained with two of the probes used in the present study to detect MOP receptor mRNA. Low power images of film autoradiograms obtained after hybridizing coronal sections from rat brain. Dark regions correspond to areas rich in hybridization signal. The same pattern of hybridization is observed when each oligonucleotide was used independently as hybridization probe in consecutive sections, A1-B1: Specific hybridization signals were not observed when a large excess of the same unlabelled oligo was present during hybridization, A2-B2. The thermal stability of the hybrids was checked by washing the hybridized tissues at increasing temperatures: serial sections were washed at 60�C (A1-B1), 70�C (A3-B3) or 80�C (A4-B4). Scale bar = 3 mm. This Figure complements Figure 1 of the main manuscript.
• Data Supplement - Supplementary Figure 2. Illustrates the predominantly intracellular location and movement of c-Myc-5-HT2A-eCFP containing vesicles following induction of expression in Flp-In T-REx HEK293 cells. The movie covers a 20 minute period and complements Figure 2A (right hand panel) of the main manuscript.
• Data Supplement - Supplementary Figure 3. Membranes were prepared from Flp-In T-REx HEK293 cells stably expressing MOP-eYFP and harboring c-Myc-5-HT2A-eCFP at the Flp-In locus that were untreated (open squares) or treated with either doxycycline (filled squares) or doxycycline plus 5-HT (10-5M) (filled diamonds). The ability of varying concentrations of either DAMGO (left hand panel) or morphine (right hand panel) to stimulate binding of 35SGTPgS was then assessed.
• Data Supplement - Supplementary Figure 4. Illustrates the time course (over a 30 minute period) of internalization of MOP-eYFP in response to DAMGO in the absence of expression of c-Myc-5-HT2A-eCFP. This complements the set of three images in the top row of the left hand section of Figure 6 of the main manuscript.
• Data Supplement - Supplementary Figure 5. Illustrates the lack of internalization of MOP-eYFP in response to morphine in the absence of expression of c-Myc-5-HT2A-eCFP. This complements the set of three images in the middle row of the left hand section of Figure 6 of the main manuscript.
• Data Supplement - Supplementary Figure 6. Illustrates the internalization of MOP-eYFP in response to a combination of morphine plus 5-HT in the presence of c-Myc-5-HT2A-eCFP. This complements the set of three images in the third row of the right hand section of Figure 7 of the main manuscript.