1% Tween 20 at room temperature for 2 hours. After extensive washing, the membranes were incubated with polyclonal goat anti-rabbit IgG antibody (1:2000 by volume) conjugated with horseradish peroxidase. The membranes were washed in PBS, and the chemiluminescent substrate was added. The membranes were stripped and stained with Coomassie Blue R-250 for verification of the EX 527 ic50 loading sample. Quantitative
RT-PCR Analysis Quantitative RT-PCR was performed to characterize the expression profile of human target genes by using the human quantitative (q) RT-PCR arrays (Origene) per the manufacturer’s instructions. Polymerase chain reaction was performed in 96-well optical plates using the iCycler (Bio-Rad Laboratories, Hercules, CA, USA) with primers specific for Prx I-VI, Trx1, Trx2, β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and iQ SYBR Green Supermix (Bio-Rad).
The resulting fluorescence proportional to the amount of amplified DNA was measured at the end of each elongation phase at 530 nm. A standard graph of CT (the point at which the fluorescence crosses the threshold) values obtained from serially diluted target genes was constructed for all reactions to ensure selleck products that they were amplified and reported in proportion to template. CT values were converted to gene copy number of the template cDNA using the equation 2ΔΔCT. The ΔCT is the abundance of cDNAs for transcripts of each gene normalized to the β-actin and GAPDH at each time point. The ΔΔCT is obtained by subtracting a calibrator value for each gene transcript almost being assayed. In parallel with each cDNA sample, standard curves were generated to correlate CT values using serial dilutions of the target gene. The quality of the standard curve was judged from the slope and the correlation coefficient. Quantification was performed by comparing the fluorescence of a PCR product of unknown concentration with the fluorescence of several dilutions. Melting curve analysis was used for product validation. The primers for β-actin and GAPDH were supplied by Origene. Other selleck screening library Primer sequences are summarized in Table 2. Table 2 Sequence of Primers for Real-Time PCR1 Amplification
Primer for Direction Primer Sequence (5′ to 3′) Human Prx I Forward tttggtatcagacccgaagc Reverse tccccatgtttgtcagtgaa Human Prx II Forward ccagacgcttgtctgaggat Reverse acgttgggcttaatcgtgtc Human Prx III Forward gttgtcgcagtctcagtgga Reverse gacgctcaaatgcttgatga Human Prx IV Forward cagctgtgatcgatggagaa Reverse taatccaggccaaatgggta Human Prx V Forward ccctggatgttccaagacac Reverse aagatggacaccagcgaatc Human Prx IV Forward cgtgtggtgtttgtttttgg Reverse tcttcttcagggatggttgg Human Trx1 Forward ctgcttttcaggaagccttg Reverse tgttggcatgcatttgactt Human Trx2 Forward agcccggacaatatacacca Reverse aatatccaccttggccatca 1 Abbreviations: PCR, polymerase chain reaction; Prx, peroxiredoxin; Trx, thioredoxin. Statistical Analysis Continuous data were reported with mean and standard error (S.E.