Melanopsin-Containing Retinal Ganglion Cells: Architecture, Projections, and Intrinsic Photosensitivity S. Hattar, H.-W. Liao, M. Takao, D. M. Berson, and K.-W. Yau

Supplementary Material

Supplemental Figure 1. Primary structure of rat melanopsin and characterization of the amino-terminal antibody. (A) Predicted amino-acid sequence of rat melanopsin. (B) Western blots of total protein extract from HEK 293 cells expressing myc-tagged rat melanopsin to verify specificity of the amino-terminal peptide antibody (Anti-MOP). Arrowhead indicates expected molecular mass of melanopsin. The band of larger molecular mass is probably glycosylated melanopsin because it disappeared in deglycosylated conditions. Mock transfection with plasmid pRK5 was used as negative control.

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METHOD: The rat melanopsin cDNA, with a myc-tag at the carboxy-terminus, was subcloned into the pPRK5 expression vector and transfected into HEK 293 cells (Clontech, Palo Alto, CA) using SuperFect Transfection Reagent (Qiagen, Valencia, CA). Two days after transfection, total proteins were extracted from the transfected cells with a proteinase inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN), 5 mM EDTA, 1% Triton X-100 and PBS. The proteins were denatured in 5% 2-mercaptoenthanol, followed by SDS-PAGE. The anti-melanopsin and anti-myc antibodies were diluted 1:1000-2000, and the secondary, horseradish peroxidase-coupled anti-rabbit IgG was diluted 1:10,000. The substrate system was ECL Plus Reagent (Amersham Life Science, Piscataway, NJ).