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. Author manuscript; available in PMC: 2013 Aug 13.
Published in final edited form as: J Neurosci. 2013 Feb 13;33(7):3178–3189. doi: 10.1523/JNEUROSCI.2428-12.2013

Figure 1. Expression screening for inhibitor of RPE65.

Figure 1

(A) The visual cycle reactions taking place in RPE. LRAT esterifies all-trans retinol (atROL) from photoreceptors and blood to all-trans retinyl esters (atRE), such as all-trans retinyl palmitate (atRP); RPE65 isomerohydrolyzes atRP to 11-cis retinol (11cROL), which is then oxidized by retinol dehydrogenase (RDH) to 11-cis retinaldehyde (11cRAL). (B) Secondary screening of 30 subpools (24 shown) from a bovine RPE expression library pool #7 (Jin et al., 2005). Amounts of 11cROL synthesized from atROL added into the media of 293T-LRC cells transfected with the indicated library pools were analyzed by HPLC. Cells transfected with subpool #3 (*) contained the least amount of 11cROL. (C) Tertiary screening of 32 subpools (27 shown) of pool #7:3. Isomerase assays were performed as in (B). Subpool #7:3:24 (*) was selected for the final screening. (D) HPLC chromatogram of retinoids extracted from the control cells transfected with pRPE65 and pRK5 (mock) plasmids. The peak corresponding to 11cROL is indicated. (E) UV spectrum acquired from the 11cROL peak in (D). (F) HPLC chromatogram of retinoids extracted from the cells transfected with subpool #7:3:24. (G) Isomerase assays of 60 single clones (40 shown) from subpool #7:3:24. Clones for ELOVL1 (e), RDH5 (r), FATP4 (f), and PSMD13 (p) are indicated.