Fig. 5.

Sgk1 stabilizes hOAT3. a. Top panel: hOAT3 expression at the cell surface. COS-7 cells were co-transfected with hOAT3 and control vector or with hOAT3 and constitutive active form of sgk1 (CA-sgk1). The transfected cells were treated with brefeldin A (BFA, 0.1 μg/ml) for 6 hrs., followed by the determination of hOAT3 expression at the cell surface through a biotinylation approach and immunoblotting with anti-myc antibody (epitope myc was tagged to hOAT3 for immune-detection). Bottom panel: The expression of cell surface marker protein E-cadherin. The same blot from Fig. 5a, Top panel was re-probed with anti- E-cadherin antibody. b. Densitometry plot of results from Fig. 5a, Top panel as well as from other experiments. The amount of cell surface hOAT3 was expressed as % of total initial cell surface hOAT3 pool (at 0 hr. of BFA treatment). Values are mean ± S.E. (n = 3). c. Top Panel: Total cell expression of hOAT3. COS-7 cells were co-transfected with hOAT3 and control vector or with hOAT3 and constitutive active form of sgk1 (CA-sgk1). The transfected cells were treated with brefeldin A (BFA, 0.1 μg/ml) for 6 hrs. Treated cells were lysed, followed by immunoblotting (IB) with an anti-myc antibody. Bottom panel: Total cell expression of β-actin, a cellular protein marker. The same blot from Fig. 5c, Top panel was re-probed with anti-β-actin antibody. d. COS-7 cells were co-transfected with hOAT3 and control vector, or with hOAT3 and CA-sgk1. The transfected cells were treated with brefeldin A (BFA, 0.1 μg/ml) for 6 hrs. [³H] estrone sulfate uptake (3min, 0.3 μM) was then measured. Uptake activity was expressed as % of the uptake value measured at 0 hr. of BFA treatment.