Figure 1.

Zebrafish Rbpr2 mediates retinol uptake in NIH3T3 cells. (A) Western blot analysis confirmed co-expression of zebrafish Rbpr2 and human LRAT proteins in stable NIH3T3 clones. (B) Subcellular localization of zebrafish Rbpr2 (V5-tagged, green) and human LRAT (red) in stable NIH3T3 cells determined by immunohistochemistry and confocal microscopy. Nucleus, stained with DAPI (blue). Scale bar = 50 μm. (C) Parental NIH3T3 cells (diamond) or NIH3T3 cells expressing LRAT only (squares) or NIH3T3 cells expressing Rbpr2 only (triangle) or NIH3T3 cells co-expressing Rbpr2+LRAT (X) were incubated with [³H]ROL-RBP4. Cells were washed thrice and lyzed at the 0, 15 and 30 min time points. Protein concentrations were estimated and cells were subjected to scintillation counting. The x-axis shows the concentration of retinol-bound RBP4 taken up by the cells and expressed as fmole/mg of protein/min. (D) Representative images of absorption spectrum of recombinant RBP4-ROL at A280/330 nm. Inset, purified recombinant RBP4 protein, resolved by SDS-PAGE and visualized by Commassie Blue staining. *p < 0.005; [³H]ROL-RBP4 uptake values in NIH3T3 cells co-expressing Rbpr2 + LRAT compared to NIH3T3 cells expressing Rbpr2 receptor only.