Ion PCR efficiencies were acquired by the amplification of order Tubastatin A dilution series of cDNA according to the equation 10(21/slope) and consistent between target mRNA and GAPDH mRNA. Negative controls were performed in which cDNA was substituted for water.Materials and Methods Animals and Sample CollectionTwenty littermates of suckling Huanjiang mini-piglets were used and nursed by primiparous gilts in the present study. The gilts were individually housed and fed a maize- and soybean mealbased diet and housed in the same pigsty [20]. On days 0, 7, 14 and 21 of age, five littermates were chosen and two piglets from per littermate (one with the largest BW and another with 12926553 the lowest BW) were sampled, respectively. Piglets were individually weighed immediately before feeding. Blood samples (about 5 ml from each piglet) were collected into 10-mL heparin-coated tubes and centrifuged at 3,0006g and 4uC for 10 min. Then, the supernatants (plasma) were stored at 220uC until required for analysis of AA content. Immediately after blood sampling, piglets held under general anaesthesia and then killed by an intravenous injection of the 4 sodium pentobarbital solution (40 mg/kg body weight) [21]. Samples of proximal jejunum (after cleaned by icecold phosphate-buffered saline), longissimus dorsi muscle, and liver were collected and immediately frozen in liquid 
nitrogen and then stored at 270uC until analysis. All the experimental procedures used in this study were approved by the Animal Care and Use Committee of Chinese Academy of Sciences [22?3].Determination of Protein Quantity of ASCT2 and B0ATThe frozen jejunum samples were powdered under liquid nitrogen, and lysed in RIPA buffer (150 mM NaCl, 1 Triton X100, 0.5 sodium deoxycholate, 0.1 SDS, 50 mM Tris-HCl at pH 7.4, plus a protease inhibitor cocktail purchased from Roche, Shanghai, China). After centrifugation at 10,0006g and 4uC for 10 min, protein concentration in the supernatant fluid was determined using the Bicinchoninic Acid assay (Beyotime Biotechnology, Haimen, China). All samples were adjusted to an equal protein concentration and then diluted with 26loading buffer (0.63 ml of 0.5 M Tris-HCl (pH 6.8), 0.42 ml 75 glycerol, 0.125 g sodium dodecyl sulfate (SDS), 0.25 ml b-mercaptoethanol, 0.2 ml 0.05 solution of bromphenol blue, and 1 ml water) to Table 1. Primers used for real-time PCR.TA1 (uC)Determination of AA Contents in Plasma, Liver and MusclePlasma AA contents were determined as previously described [24?5]. In brief, 1 ml of the plasma sample and 2.5 ml of 7.5 trichloracetic acid solution were mixed thoroughly and centrifuged at 12,0006g and 4uC for 15 min. The supernatant fluid was collected for analysis of AA by an ion-exchange AA analyzer 
(Tokyo, Japan). To measure the 15755315 contents of AA in the muscle and liver, about 0.1 g freeze-dried muscle or liver tissue was ground and hydrolyzed in 10 mL of 6 mol/L HCl at 110uC for 24 h. The solution was then adjusted to the volume of 100 mL and then a 1 mL of the settled solution was used for further analysis after a 10-fold dilution. Plasma samples were Felypressin manufacturer filtered through a 0.45 mm membrane before analysis [26].Genes Slc1aPrimers Forward ReverseSequences (59-39) GATTGTGGAGATGGAGGATGTGG TGCGAGTGAAGAGGAAGTAGATGA GA TCTGTCCACAACAACTGCGAG CAGCGAAGTTCTCCTGCGTC AAGGAGTAAGAGCCCCTGGA TCTGGGATGGAAACTGGAASize (bp)Slc6a19 Forward Reverse GAPDH Forward Reverse1 TA, Annealing temperature. doi:10.1371/journal.pone.0050921.tNeutral Amino Acids in Mini-Pigletsa.Ion PCR efficiencies were acquired by the amplification of dilution series of cDNA according to the equation 10(21/slope) and consistent between target mRNA and GAPDH mRNA. Negative controls were performed in which cDNA was substituted for water.Materials and Methods Animals and Sample CollectionTwenty littermates of suckling Huanjiang mini-piglets were used and nursed by primiparous gilts in the present study. The gilts were individually housed and fed a maize- and soybean mealbased diet and housed in the same pigsty [20]. On days 0, 7, 14 and 21 of age, five littermates were chosen and two piglets from per littermate (one with the largest BW and another with 12926553 the lowest BW) were sampled, respectively. Piglets were individually weighed immediately before feeding. Blood samples (about 5 ml from each piglet) were collected into 10-mL heparin-coated tubes and centrifuged at 3,0006g and 4uC for 10 min. Then, the supernatants (plasma) were stored at 220uC until required for analysis of AA content. Immediately after blood sampling, piglets held under general anaesthesia and then killed by an intravenous injection of the 4 sodium pentobarbital solution (40 mg/kg body weight) [21]. Samples of proximal jejunum (after cleaned by icecold phosphate-buffered saline), longissimus dorsi muscle, and liver were collected and immediately frozen in liquid nitrogen and then stored at 270uC until analysis. All the experimental procedures used in this study were approved by the Animal Care and Use Committee of Chinese Academy of Sciences [22?3].Determination of Protein Quantity of ASCT2 and B0ATThe frozen jejunum samples were powdered under liquid nitrogen, and lysed in RIPA buffer (150 mM NaCl, 1 Triton X100, 0.5 sodium deoxycholate, 0.1 SDS, 50 mM Tris-HCl at pH 7.4, plus a protease inhibitor cocktail purchased from Roche, Shanghai, China). After centrifugation at 10,0006g and 4uC for 10 min, protein concentration in the supernatant fluid was determined using the Bicinchoninic Acid assay (Beyotime Biotechnology, Haimen, China). All samples were adjusted to an equal protein concentration and then diluted with 26loading buffer (0.63 ml of 0.5 M Tris-HCl (pH 6.8), 0.42 ml 75 glycerol, 0.125 g sodium dodecyl sulfate (SDS), 0.25 ml b-mercaptoethanol, 0.2 ml 0.05 solution of bromphenol blue, and 1 ml water) to Table 1. Primers used for real-time PCR.TA1 (uC)Determination of AA Contents in Plasma, Liver and MusclePlasma AA contents were determined as previously described [24?5]. In brief, 1 ml of the plasma sample and 2.5 ml of 7.5 trichloracetic acid solution were mixed thoroughly and centrifuged at 12,0006g and 4uC for 15 min. The supernatant fluid was collected for analysis of AA by an ion-exchange AA analyzer (Tokyo, Japan). To measure the 15755315 contents of AA in the muscle and liver, about 0.1 g freeze-dried muscle or liver tissue was ground and hydrolyzed in 10 mL of 6 mol/L HCl at 110uC for 24 h. The solution was then adjusted to the volume of 100 mL and then a 1 mL of the settled solution was used for further analysis after a 10-fold dilution. Plasma samples were filtered through a 0.45 mm membrane before analysis [26].Genes Slc1aPrimers Forward ReverseSequences (59-39) GATTGTGGAGATGGAGGATGTGG TGCGAGTGAAGAGGAAGTAGATGA GA TCTGTCCACAACAACTGCGAG CAGCGAAGTTCTCCTGCGTC AAGGAGTAAGAGCCCCTGGA TCTGGGATGGAAACTGGAASize (bp)Slc6a19 Forward Reverse GAPDH Forward Reverse1 TA, Annealing temperature. doi:10.1371/journal.pone.0050921.tNeutral Amino Acids in Mini-Pigletsa.