Volume 119, Issue 6, Pages 1623-1630 (December 2000) Nutritional regulation of nucleoside transporter expression in rat small intestine  Raquel Valdés, *, María A. Ortega, ‡, F.Javier Casado, *, Antonio Felipe, *, Angel Gil, ‡, Antonio Sánchez–Pozo, ‡, Marçal Pastor– Anglada, *  Gastroenterology  Volume 119, Issue 6, Pages 1623-1630 (December 2000) DOI: 10.1053/gast.2000.20183 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Characterization of anti-CNT2 (SPNT) antiserum. (A) Western blot analysis of homogenates (H) and plasma membrane preparations (PMV) from rat liver, by using the preimmune and immune antisera from the same rabbit. A representative blot (20 μg protein loaded in each lane). (B) Lack of cross-reactivity between the 2 oligopeptides used to obtain the anti-CNT1 and anti-CNT2 (SPNT) antisera. Twenty micrograms of the uncoupled oligopeptide was run on a 15% polyacrylamide gel. The oligopeptides were then detected by Western blot by using the anti-CNT1 and anti-CNT2 (SPNT) antisera. Gastroenterology 2000 119, 1623-1630DOI: (10.1053/gast.2000.20183) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Tissue distribution of CNT1 and CNT2 proteins. The monospecific antibodies against the CNT1 and CNT2 isoforms specifically recognize single bands in the 55–60-kilodalton range. A representative Western blot analysis, using either immune antiserum or prebleed serum from the same rabbit. Equal amounts of protein were loaded (20 μg) to allow direct comparisons of CNT levels among rat tissues. L, liver; K, kidney; Lu, lung; P, pancreas; Br, brain; M, skeletal muscle; H, heart; B, brown adipose tissue; I, small intestine. Gastroenterology 2000 119, 1623-1630DOI: (10.1053/gast.2000.20183) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of fasting on CNT1 protein tissue levels. (A) A representative Western blot of homogenates from selected tissues. Two independent samples from each experimental condition (fed and 48-hour starved rats) were run. Ten micrograms of protein was loaded for liver, small intestine, and kidney homogenates; 20 μg protein was used for heart and brown adipose tissue Western blot. (B) Densitometric analysis of CNT1 amounts in liver (L), small intestine (I), kidney (K), heart (H), and brown adipose tissue (B). □, Fed rats; ■, fasted rats. Results are the mean ± SEM of 4 independent samples. Gastroenterology 2000 119, 1623-1630DOI: (10.1053/gast.2000.20183) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Characterization of brush border membrane vesicles from 48-hour starved and fed rat jejunum. Plasma membrane vesicle preparations from rat jejunum were checked for their apical origin by monitoring the amount of the rBAT protein, an amino acid transporter known to be expressed at the apical side of the enterocyte. CNT1 protein amounts were also analyzed in the same samples. A representative Western blot is shown. H, homogenate; V, brush border plasma membrane vesicles. Gastroenterology 2000 119, 1623-1630DOI: (10.1053/gast.2000.20183) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Thymidine and gemcitabine uptake into brush border plasma membrane vesicles from 48-hour starved (■) and fed (□) rat jejuni. One-micromolar [3H]thymidine and [3H]gemcitabine uptakes into brush border plasma membrane vesicles from fed and 48-hour starved rats were measured by rapid filtration (see Materials and Methods), by using either an NaSCN or a KSCN medium. The Na+-dependent fraction of thymidine and gemcitabine uptake was calculated by substracting the uptake rates measured in the K+ medium from those determined in the Na+ medium. Results are the mean ± SEM of quintuplicate measurements made on 4 independent rat plasma membrane vesicle preparations. Gastroenterology 2000 119, 1623-1630DOI: (10.1053/gast.2000.20183) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 CNT1 expression in jejunum and liver from rats fed N+ and N− diets. (A) A representative Western blot of homogenates from liver and jejunum. Two independent samples from each experimental condition (animals fed N+ and N−) were run. (B) Densitometric analysis of CNT1 amounts in liver (L) and jejunum (I). □, N− diet; ■, N+ diet. Results are the mean ± SEM of 4 independent samples. Gastroenterology 2000 119, 1623-1630DOI: (10.1053/gast.2000.20183) Copyright © 2000 American Gastroenterological Association Terms and Conditions