Figure S3 Identification of CHO stably expressing MOR cell lines and CHO stably expressing MOR and β-arrestin2 cell lines.
  1. Identification of MOR expression of CHO-MOR(WT & mutant) cell lines by western-blot analysis. Cells were lysed and immunoblotted with the anti-MOR antibody. To ensure equal loading into each lane, blots were also incubated with GAPDH antibody.
  2. Identification of MOR mutation of CHO-MOR (WT & mutant) cell lines by PCR analysis. Genomic DNA was extracted from each CHO-MOR (WT & mutant) cell line, MOR DNA fragments were amplified from genomic DNA by PCR, then the mutant sites of MOR were confirmed by DNA sequencing.
  3. Identification of β-arrestin2 expression of CHO-MOR (WT & mutant)-β-arresin2 cell lines by western-blot analysis. Cells were lysed and immunoblotted with the anti-β-arrestin2 antibody. To ensure equal loading into each lane, blots were also incubated with GAPDH antibody.
  4. Identification of MOR expression on the membrane of CHO-MOR (WT & mutant) cell lines by immunofluorescence analysis. MOR on the cell membrane can be labeled with SNAP-Lumi4-Tb through incubation, then the expression level of MOR-Tb was read on Envision Multilabel Reader at 615 nm. The data are presented as mean ± SEM of three assays run in duplicate.