Figure 5
From: The dual roles of RPE65 S-palmitoylation in membrane association and visual cycle function
Palmitoylation levels and isomerisation activity of RPE65 wild type and cysteine mutants in in vitro minimal visual cycle system. (A) HEK293F cell cultures transfected with pVitro2/RPE65 wild type or cysteine mutants + CRALBP and pVitro3/LRAT or LRATC161S mutant + RDH5 vectors were incubated with 2.5 μM all-trans retinol (at-ROL) for 7 h. Cell extracts were analysed by acyl-RAC assay to monitor changes in the palmitoylation status of RPE65. Samples were treated with 0.5 M hydroxylamine (HAM; indicated as “+”) or 0.5 M NaCl (indicated as “−”), respectively. (B) In vitro isomerase activity was assessed for wildtype RPE65 and cysteine mutants (C112, C146 and C195). Data were represented as mean ±S.D. from three independent experiments. *P < 0.01, **P < 0.05, ***P < 0.0005 and ****P < 0.0001 unpaired and paired student’s t-test.
