Rhodopsin / Forward / 5' TCC TGA TCT GCT GGC TTC CC TACG 3'
Reverse / 5' CGC AAC TGT ATG CTC ACC ACG CTG 3'
Melanopsin / Forward / 5' ACC CAC CGC TCC ACA TTG 3'
Reverse / 5' CAG CCC ACT TCA CTC TCA GAA 3'
GRK2 / Forward / 5' AGC GAG TAC CCA AGA TGA AGA ACA 3'
Reverse / 5' CAC TGC CAC GCT GGA TCA 3'
GRK3 / Forward / 5' GGA CAG AAG TCG TTA CAG CTA ATT CA 3'
Reverse / 5' GCC ACT TCT CTT TAA ATA ACA AAC TAT 3'
GRK4 / Forward / 5' TCA GAC CAA AGA GAA ACT TCT TCC3'
Reverse / 5' CTC ACT GAG GGC AAT GTT CA 3'
GRK5 / Forward / 5' AGG CCG AGT CCA TCT GCA ACA TGC TG 3'
Reverse / 5' GGC TCT GAG CTG GTT CCA CTT TTG AC 3'
GRK6 / Forward / 5' GCC TGA TCC CCA GGC TAT TTA TTG C 3'
Reverse / 5' GGG ACG CAC CTT TGT CCC AAG 3'
Supplemental Table 1. List of primer sequences
Supplemental Figure 1.
S1. Membrane preperations containing ~ 250 nmol of mouse melanopsin or phosphonull mutanta melanopsin were resuspended in kinase buffer andreconstituted with 50 µM 11-cis retinal. Samples were either exposed to white light for 30 min, or left in the dark. Audioradiogram of reactions run on a 10% SDS polyacrylamide gel dried down and placed on X-ray film.
S2. Antibody control. HEK 293 cells transfected with wild type melanopsin, phosphonull melanopsin or a truncation mutant of melanopsin 386, which ends after amino acid 385 upstream of the C-terminal antibody epitope sequence. Cells were seeded onto cover slips twenty-four hours post transfection. Cells were fixed in 4% PFA for thirty minutes twenty-four hours after seeding. Cells were blocked and solubilized in PBS supplemented with 10% normal goat serum and 0.3% tween-80 for one hour. Cells were incubated in either anti-N-terminal (1:1000 dilution) or anti-C-terminal antibody (Thermo Scientific, 1:100 dilution) overnight in a humidified chamber at room temperature. Cells were washed three times in PBS-T and incubated with alexafluor 488 anti-rabbit secondary antibody for 1 hour. Cells were washed as above and mounted on a slide with mounting media containing the nuclear stain DAPI. Images were taken with Lecia SP5 confocal microscope at 60X magnification. Antibody staining is shown in green and blue staining corresponds to the DAPI nuclear stain. Staining shows all cells transfected wit any of the melanopsin constructs are labeled with the N-terminal antibody, while only the wild type and phosphonull transfected cells react with the C-terminal antibody.