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	Fig 2. Plasma membrane localization of hENT1 variants.Fluorescent confocal microscope images of WT hENT1, ∆EL1, ∆IL6, and ∆EL1/∆IL6 loop deletion mutants, T248 and S254A point mutants, and ∆1CTM and ∆3CTM carboxyl-terminus deletion mutant expressed in X. laevis oocytes. Constructs are indicated on the panels. The individual images along the focal plane of the microvilli on the membrane (EGFP in green, mch-TMEM in red) are shown, followed by the merge of EGFP and mch-TMEM in (yellow). Orthogonal sections through Z stacks also show co-localization. Carboxyl-terminal deletions did not show EGFP fluorescence on the membrane. Images are representative of 30 oocytes. Scale bar = 5μm.
	Fig 3. Intracellular distribution of the hENT1 variants.Shown are the confocal images for WT hENT1, ∆EL1, and ∆EL1/∆IL6 loop deletion mutants, S254A point mutant, ∆3CTM carboxyl-terminus deletion mutant expressed in X. laevis oocytes. Two images were taken: one on the plasma membrane (left panel) and the other deep within the cell showing the ER structure (right panel). Only the orthogonal sections are shown for ∆IL6, T248A and ∆1CTM. Left panel shows normal distribution of EGFP (green) on the membrane and mch-KDEL (red) in the ER for the WT, T248A and S254A. In contrast, ∆EL1, ∆IL6, ∆EL1/∆IL6 shows some co-localization of hENT1 and KDEL in the ER. Carboxyl-terminal deletions did not show EGFP fluorescence on the membrane. Instead, EGFP was present within the ER, co-localized with mch-KDEL. All the confocal images show a single, representative, section of a Z-series taken through the entire cell. Images are representative of 30 oocytes. Scale bar = 5μm.
	Fig 4. hENT1 loop deletion mutants show similar sub-cellular distribution and susceptibility to NBMPR inhibition.(a) Fraction of intra- and extra-cellular distribution of EGFP-hENT1 variants. Confocal images shown in Fig 2 were quantified and the fluorescence in the ER and plasma membrane expressed as percent of the total fluorescence. Shown are means and SE for 30 oocytes. * p<0.05. The black and grey bars represent the extra and intracellular fluorescence respectively. (b) The total protein expression assessed by Western blot. 100 μg of proteins were loaded in each lane. The EGFP-hENT1 fusion proteins were visualized using EGFP monoclonal antibody. Constructs are shown above the lanes. (c) Western blots showing deglycosylation of the WT and ∆IL6 mutant. 100 μg of the proteins were incubated with and without deglycosylase for 4 hr at 37°C. (d) Western blots for carboxyl terminal deletions. 400 μg of the WT, ∆3CTM and ∆1CTM deletions were loaded in each lane and visualized with EGFP monoclonal antibody. The migration positions of the molecular weights standards are indicated in panel’s b-d. Actin was used as the internal control to show equal loading in lanes b and d. (e) Quantification of NBMPR binding sites. Oocytes were injected EGFP-hENT1 RNA were incubated for 48 hrs prior to addition of 500 nM 3H NBMPR for one hr. Radioactivity retained by washed oocytes was measured and corrected for background. Values are expressed as mean ± SE for 5 oocytes for 3 independent experiments done in triplicate. (f) Adenosine uptake after NBMPR inhibition. Values corrected for background are expressed as mean ± SE for 5 oocytes for 3 independent experiments done in triplicate. Shown are WT (closed circles), ∆IL1 (open circles), ∆IL6 (closed squares), and ∆EL1/∆IL6 (open squares).
	Fig 5. hENT1 deletion mutants show similar substrate uptake.Time dependence of uptake of 5 μM adenosine (a) and uridine (b). Concentration dependence of adenosine (c) and uridine (d) uptake. The incubation time was 1 minute for adenosine and 3 minutes for uridine. Values are corrected for background and expressed as means ± SE for 5 oocytes for 3 independent experiments done in triplicate. Shown are WT (closed circles), ∆EL1 (open circles), ∆IL6 (closed squares), and ∆EL1/∆IL6 (open squares).
	Fig 6. Effect of TBB on the deletion constructs.(a) Uptake of 5 μM 14C-adenosine after 1 minute incubation period without (black) and with (gray) pre-incubation with TBB. Values are expressed as mean ± SE for 5 oocytes for 3 independent experiments done in triplicate. (b) Western blot of the oocytes with and without treatment with TBB probed with EGFP monoclonal antibody. (c) Confocal images of oocytes co-injected with RNA expressing WT EGFP-hENT1 and mch-TMEM after treatment with 25 μM TBB for 12 hr. (d) Confocal images of oocytes co-injected with RNA expressing WT EGFP-hENT1 and mch-KDEL. Images were taken along the plane of the microvilli (left) and within the ER (right).
	Fig 1. Structure of hENT1 with deletions and mutations.Shown is the predicted topology of hENT1 with the cytoplasmic amino-terminus, 11 TMs and short extracellular carboxyl-terminus. Red bars show the beginnings and the ends of the deleted regions in the extracellular, ∆EL1, and intracellular, ∆IL6, loops and the beginnings of the deletions at the carboxyl-terminus, ∆3CTM and ∆1CTM. The predicted CK2 phosphorylation sites, Thr 248 and Ser 254, are marked in magenta. EGFP was fused to the amino-terminus.

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