Transfected HEK cells were maintained at 37℃ and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing up to 10% fetal bovine serum (FBS). Media was collected 48 hours following transfection. Cell lysates from HEK-293 cells expressing N40D MOR N-terminal peptides were collected and centrifugation was performed to remove all non-soluble components from the cell culture media. 5% SDS and 1 M DTT were added to the lysate and heated to 95℃ for 5 minutes to denature total proteins contained within the soluble fraction. Denatured proteins were incubated with recombinant PNGase F (2.5 U/μL) overnight at 37℃. To stop the reactions, 4x SDS-PAGE loading buffer was added to each sample and heated at 95℃ for 5 minutes. Samples were analyzed with Western blotting using anti-FLAG tag antibody (1:2500) (Sigma Aldrich; F3165) and horseradish peroxidase-conjugated anti-mouse secondary antibody (1:20,000) (Life Technologies; 31430).
