Radiolabeled Retinol Uptake Assay

RBP4 cDNA cloned into the pET3a bacterial expression vector was used to express RBP4 in E.Coli as previously described (Shi et al., 2017 (link)). Apo-RBP4 (100 μg) was loaded with retinol in 0.2 mL of PBS by the addition of 100 μm radiolabeled retinol (American Radiolabeled Chemicals; vitamin A alcohol [3H(N)] Retinol-labeled, adjusted to 1 μCi/nmol specific activity by the addition of cold retinol) and incubating for 1 h at room temperature and then overnight at 4°C in light-protected tubes, as previously described (Shi et al., 2017 (link)). Stable NIH3T3 cells expressing either, RBPR2 or RBPR2 and LRAT were plated in 10 cm dishes. Cells were grown to 70% confluence, washed thrice with 1x PBS and incubated for 1 h in serum-free medium, at which point [³H]ROL-RBP4 was added for 60 min. Cells were washed thrice with 1x PBS and lysed in PBS containing 1% Nonidet P-40. Lysates were homogenized and transferred to scintillation tubes for scintillation counting. Parental NIH3T3 incubated with [³H]ROL-RBP4 served as controls. The RBP4-ROL binding and uptake assay was repeated thrice, using stable cells from a different passage.