Tween-20 with 5% skim milk, blots were probed with primary antibodies at dilutions of 1:500 and 1:1,000 overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies ( 31340 and 31455). Protein bands were visualized using the enhanced chemiluminescence system and densitometric analysis was performed using Scion Image-Release Beta 4.02 software. 1.6 qPCR assays. Primer Express software (Applied Biosystems, Foster City, CA, USA) was used to design oligonucleotide primers. PCR assays were performed in 96-well optical reaction plates using an ABI 7500HT thermal cycler (Applied Biosystems) as described previously (). Briefly, total RNA was extracted from liver and ileum tissues using Trizol reagent, and 1 µg of RNA reverse transcribed into cDNA using the primers indicated in A 25-µL reaction containing 4 µg of cDNA, 12.5 µL Table. S1. of SYBR Master mix, and 1 µL of each primer was subjected to thermal cycling, involving an initial denaturation step at 95°C for 10 min and 45 cycles of for 15 s, 60°C for 1 min). All reactions were (95°C performed in triplicate, and the averages of the threshold cycle (Ct) were used to interpolate curves using 7300 System SDS Software. Results are presented as the ratio of each mRNA compared with the expression level of glyceraldehyde 3-phosphate dehydrogenase mRNA. 1.5 Determination of enzymatic activity of hepatic FMO.
Tween-20 with 5% skim milk, blots were probed with primary antibodies at dilutions of 1:500 and 1:1,000 overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies ( 31340 and 31455). Protein bands were visualized using the enhanced chemiluminescence system and densitometric analysis was performed using Scion Image-Release Beta 4.02 software. 1.6 qPCR assays. Primer Express software (Applied Biosystems, Foster City, CA, USA) was used to design oligonucleotide primers. PCR assays were performed in 96-well optical reaction plates using an ABI 7500HT thermal cycler (Applied Biosystems) as described previously (). Briefly, total RNA was extracted from liver and ileum tissues using Trizol reagent, and 1 µg of RNA reverse transcribed into cDNA using the primers indicated in Table. S1. A 25-µL reaction containing 4 µg of cDNA, 12.5 µL of SYBR Master mix, and 1 µL of each primer was subjected to thermal cycling, involving an initial denaturation step at 95°C for 10 min and 45 cycles of (95°C for 15 s, 60°C for 1 min). All reactions were performed in triplicate, and the averages of the threshold cycle (Ct) were used to interpolate curves using 7300 System SDS Software. Results are presented as the ratio of each mRNA compared with the expression level of glyceraldehyde 3-phosphate dehydrogenase mRNA. 1.5 Determination of enzymatic activity of hepatic FMO.