L-DOPA Is an Endogenous Ligand for OA1
Figure 1
(A–C) Western blot analysis of proteins bound (B) or unbound (U) to strepavidin-conjugated beads after biotinylation of RPE in situ, cultured RPE (B), or COS cells transfected to express OA1-GFP (C). Blots were probed to visualize OA1 and actin after cell surface biotinylation and fractionation using strepavidin-conjugated beads. For cultured cells (B and C), cells were either maintained in 500 μM (normal DMEM) or 1 μM tyrosine for 3 d prior to analysis.
(D) Quantification of western blot analysis by densitometry. OA1 densitometry is shown as the percentage of the control for paired cell cultures, transfected, and then split into two equal groups, one of which was the control, maintained in normal DMEM (control; open bars). The other group was maintained in 1 μM tyrosine DMEM (LT; solid bars) until harvest. Paired t-test analysis was used to test whether the difference was significant, and an asterisk (*) denotes p < 0.001. Actin, analyzed the same way, showed no differences, and p = 0.724.
(E and F) Composite confocal microscopy of pigmenting RPE cells maintained in normal DMEM (E) or 1 μM tyrosine (F), and then stained with anti-OA1 antibodies and imaged at 20×. Bar represents 25 μm.