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. 2015 Apr 29;56(4):2553–2567. doi: 10.1167/iovs.14-16298

Figure 1.

Figure 1

High-throughput screening and orthogonal assays for the translocation and clearance of P23H mutant opsin in mammalian cells. (A) The β-Gal fragment complementation assay applied to a HTS for P23H mutant opsin translocation. Expressed in a U2OS cell line, the large subunit of β-Gal (EA, green tag) was fused with a plasma membrane anchored peptide, PLC (orange), whereas the small subunit of β-Gal (PK, pink tag) was fused on the C-terminal of P23H mutant opsin (blue). Only when P23H mutant opsin is transported to the PM upon treatment with an active compound will β-Gal be reconstituted and its restored activity quantified by its luminescence. (B) Diagram of the BRET assay for confirmation of P23H mutant opsin translocation. Briefly, the translocation of P23H mutant opsin was read by a BRET signal (dual emission ratio 530/480 nm) from a double stable cell line (HEK293) coexpressing mouse P23H mutant opsin (blue) fused with Renilla luciferase (Rluc, green) as an energy donor and membrane associated Kras peptide (orange) conjugated with fluorescent protein Venus (pink) as an acceptor. Increased PM transport of P23H-Rluc will increase the BRET signal. (C) The HTS assay for clearance of P23H mutant opsin. P23H mutant opsin was fused with Rluc and expressed in HEK293 cells to generate a stable cell line for the HTS assay. Treatment with an active compound will decrease P23H-Rluc levels noted as a decrease in luminescence from a microplate reader. (D) The amount of P23H mutant opsin expressed in a NIH3T3 stable cell line was quantified by the ALPHA immunoassay. Briefly, cells were fixed and lysed for exposure of epitopes of P23H mutant opsin to be captured by the biotinylated B6-30 anti-rhodopsin antibody and the 1D4 anti-rhodopsin antibody conjugated with acceptor beads. Upon addition of streptavidin-conjugated donor beads that bind to the biotinylated B6-30 antibody, the donor and acceptor beads are brought in close proximity. The laser excited donor (680 nm) bead then releases a singlet oxygen molecule and initiates an energy transfer to the nearby acceptor bead, which emits a red luminescence at 615 nm. Treated with active compounds, the reduced amount of P23H mutant opsin is represented by a lowered ALPHA signal.