ELISA internalization assays

CHO cells expressing Flag-epitope tagged KOR (CHO-KOR cells) or PC12 cells stably expressing SpH-KOR (SpH-KOR cells) were seeded in complete growth media into 24-well plates (2 × 10⁵ cells per well). Next day, cells were rinsed with PBS followed by labeling with mouse anti-Flag antibodies for CHO-KOR cells or chicken anti-GFP antibodies for SpH-KOR cells (1:1000 in PBS containing 1% BSA) for 1 hr at 4°C, followed by treatment with 0–10 μM of Dyn A or Dyn B in growth media containing protease inhibitor cocktail (Sigma-Aldrich; Cat. No. P2714) for 60 min at 37°C. Cells were briefly fixed (3 min) with 4% paraformaldehyde followed by three washes (5 min each) with PBS, and incubation with anti-mouse or anti-chicken antibody coupled with horse-radish peroxidase (1:1000 in PBS containing 1% BSA) for 90 min at 37°C. Cells were washed three times with 1% BSA in PBS (5 min each wash), and color was developed by the addition of the substrate o-phenylenediamine (5 mg/10 ml in 0.15 M citrate buffer [pH 5] containing 15 μl of H₂O₂). Absorbance at 490 nm was measured with a Bio-Rad ELISA reader. Values obtained with secondary antibody in the absence of primary antibody were taken as non-specific and subtracted from all points. The percentage of internalized receptors was calculated by taking total cell surface receptors before agonist treatment for each individual experiment as 100% and subtracting percent surface receptors following agonist treatment. Data presented are mean ± SE of three independent experiments in triplicate.