Figure S3 Identification
of CHO stably expressing MOR cell lines and CHO stably expressing MOR
and β-arrestin2 cell lines.
- 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.
- 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.
- 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.
- 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.