Experimental design of label-free btOCT1 binding to PGE2 by SPR.

<p><b>a</b>, Experimental design of the SPR kinetic studies of btOCT1 binding to PGE2. Control compound PEG11-Biotin and test compound PGE2-PEG11-Biotin were immobilized on to two separate sample flow cells (3 and 4) with 3-D Streptavidin (SA) hydrogel, respectively. Blocking of the inactive sites on the flow cell surfaces using biotin and subsequent stabilization of the surface using sample buffer were performed prior to analyte injection. A 2X2 fluidics mode allows btOCT1 analyte to go through the reference flow cells (1 and 2) first before sample flow cells (3 and 4). <b>b</b>, BtOCT1 binds to immobilized PGE2 specifically. Steady-state response of btOCT1 as analyte binding to PGE2-PEG11-Biotin or PEG11-Biotin immobilized SA surfaces in parallel were plotted at closed squares and closed triangles. The k value of the fitted Hill function (red line) is 89 nM btOCT1.</p

OCT1 and natural metabolites interactions characterized by various studies.

<p>OCT1 and natural metabolites interactions characterized by various studies.</p

Purified btOCT1 binds to fluorescently labeled Prostaglandin E2 (PGE2-Rhodamine) with submicromolar affinity.

<p>Black closed squares are experimental data with error bar showing standard deviation between 3 independent measurements of separately prepared btOCT1 samples. Red line shows the fitted curve (Hill function) with k value = 5.404E-7 ±5.694E-8 M, representing the K_D/affinity of btOCT1 towards the substrate PGE2. <b>Insert α</b>, shows rhodamine 123, the fluorophore of the PGE2-Rhodamine conjugate, does not show binding to similarly prepared btOCT1 protein. <b>Insert β</b> shows that the detergent used to purify btOCT1 has no consequence on the FP measurement of rhodamine 123 in the condition where the measurements were taken.</p

SPR kinetics of label-free btOCT1 binding to streptavidin-immobilized PGE2-PEG11-Biotin revealed a slow association and dissociation rates, as well as a binding affinity of approximately 100 nM.

<p><b>a</b>, Experimental design of the SPR kinetic studies of btOCT1 binding to PGE2. For a 2-over-2 kinetic analysis using samples from the same preparation, both sample flow cells (3 and 4) were loaded with PGE2-PEG11-Biotin, with different concentrations to achieve different ligand density. <b>b.</b> Different ligand density on two sample flow cells (3 and 4) was achieved by using 30 nM and 40 nM, respectively, of PGE2-PEG11-Biotin during ligand loading. <b>c</b> and <b>d</b>, Two concentration series of btOCT1 as analytes binding to two surface ligand densities of PGE2-PEG11-Biotin immobilized via SA surfaces. The two parallel kinetic analyses yielded comparable K_D of approximately 100 nM for PGE2, with an association constant of 4837 M^-1s^-1 and dissociation constant of 4.69E-4 s^-1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152969#pone.0152969.t001" target="_blank">Table 1</a>). Red lines are fitted binding kinetic curves.</p

Purified native btOCT1 is greater than 99% pure.

<p><b>a</b>. Typical size exclusion chromatography (SEC) chromatogram of affinity-purified and post-ultracentrifugation btOCT1. Red lines mark chromatography fractions (0.25 mL each) collected around btOCT1 peaks. Indicated in blue bracket are fractions having higher molecular weight (HMW) species in the Flag-affinity purified btOCT1 post ultracentrifugation pool. Monomeric fractions (indicated by red bracket) is of btOCT1 subjected to subsequent tag removal. Later elution peaks (no fraction collected) are contaminants and Flag peptides, respectively. <b>b</b>, Shows efficient expression-purification tag removal under mild conditions and the production of highly pure native btOCT1 as end product. The Flag-SumoStar tag was removed by SumoStar Protease 1 digestion after affinity and SEC chromatography at 4°C overnight in GF buffer. Closed red arrow indicates monomeric tagged btOCT1, asterisks oligomeric tagged btOCT1, closed red arrow heads monomeric tag-free btOCT1, open red arrowheads oligomeric tag-free btOCT1, red star Flag-SumoStar tag. M, molecular weight marker.</p

Development of epithelial characteristics and nephron segmentation.

<p>(A) A diagram showing the morphological progression of nephron development ex vivo, and the corresponding culture time. B) Immunofluorescent image of a 2 day (early) nephron culture. The tight junction marker ZO-1 is shown in red, and the adherens junction and marker E-cadherin is shown in green (panels B-D). C) Immunofluorescent image of 3 day (early) nephron culture. D) Immunofluorescent image of the convoluted late tubule (5 days and beyond). E) Immunofluorescent image of a 5 day (late) nephron culture. Peanut lectin marks developing podocytes (red), DAPI (blue). The late nephron begins segment specific differentiation. Bar is 50 µm (B, C, E) or 100 µm (D).</p

In silico promoter analysis of Oat1, Oat3, and Oct1 transporters predicts regulation by HNF4α.

<p>Diagram of the promoters of the human, rat, and mouse Oct1, Oat1 and Oat3 promoters. Under stringent settings only a single TRANSFAC binding motif remained - V$NR2F. Six transcripts had a DR-1 binding element within the proximal promoter region, and one had a DR-2 binding element, resulting in 7 of 9 transcripts predicted to have the potential to recruit Hnf4α directly. Green markers depict binding sites for members of the Nuclear Receptor 2 family as a function of position relevant to known transcription start sites. DR-1 elements are circled, the single DR-2 element is marked by a square. Yellow shading denotes a direct call of HNF4α by Genomatix.</p

Hepatocyte nuclear factors are associated with Slc22 genes in silico and in vitro.

<p>Evidence from the literature demonstrates the potential for functional regulation of the SLC22 family of genes by hepatocyte nuclear factors.</p

ChIP-qPCR confirms Hnf4α binds the proximal promoters of transporters in the in vivo maturing nephron.

<p>A) ChIP qPCR of Oct1 (Slc22a1), Oat1 (Slc22a6) and Oat3 (Slc22a8) promoters using an HNF4α antibody. Two loci outside of annotated coding or transcribed regions on chromosome 1 and 4 were used as negative controls. B) Schematic workflow of the chromatin immunoprecipitation qPCR experiment.</p

Informatic analysis of cultured nephrons highlights global stages of nephron development.

<p>A) self organizing map (SOM) featuring meta-meta representations of four points in nephron culture. Increasing distance between the samples on the map corresponds to increasing difference in the abstracted transcriptome. B) Non-negative matrix factorization (NMF) of the samples represented in panel A. The arrow highlights metagene F1, which comprises genes highly expressed only after 5 days (120 hours) in culture.</p