2.3. MOR internalization

Neuro2A murine neuroblastoma cells (N2A cells) stably transfected with the rat MOR cDNA epitope-tagged with HA at N-terminus (N2A-HA-rMOR) were established previously (Obeng, et al., 2019) and clones H38 and H16 expressing MOR at 1-2 pmole/mg protein were used in the study. Cells were cultured in 10-cm dishes at 37°C with 5% CO₂ in humidified air in MEM (Minimum Essential Medium, ref 41500, Gibco, NY) supplemented with 10% FBS and penicillin, streptomycin and amphotericin (A5955, Sigma, MO) and grew to 80% confluence.

MOR internalization experiments were conducted as described previously (Obeng, et al., 2019) Briefly, N2A-HA-rMOR cells were sub-cultured onto coverslips placed in 6-well plates at 300,000 cells per well. Forty-eight h later, mouse anti-HA.11 antibodies (Clone 16B12, BioLegend, CA) were added at 1:1000 to cell medium and incubated for 1 h. NFP (final 10 μM) or vehicle was added and incubated for 15 min followed by addition of etorphine (final 10 μM) or vehicle and incubated for another 15 min. Cells were cooled on ice, washed, and fixed with 4% paraformaldehyde (PFA) in PB for 15 min, and washed. Cells were then incubated with Alexa Fluor594 goat anti-mouse IgG (1:1000) (A11005, Invitrogen, OR) on a shaker with light protection for 2 h at room temperature. After washing, the cells on cover slips were mounted on slides using mounting medium (H-1200, Vector Laboratories, CA) and cured overnight. The images were acquired by a Nikon Eclipse TE300 fluoresce microscope coupled to a digital camera (MagnaFire) using 20X objective and processed with ImageJ.