Membranes were then subjected to incubation with AP conjugated to goat anti-rabbit IgG (Bio-Rad), washed, and finally developed using the AP Conjugate Substrate Kit (Bio-Rad). The multiplier of the highest dilution of the sample that, when visually assessed, gave an apparently positive reaction was defined as the amount of M protein. Finally, the amount of M protein in each sample was expressed as the mean of the results obtained
in assays performed in triplicate. For example, when a sample showed the highest positive reaction on 23 of the 2-fold dilutions (21, 22, 23, 24, and so on) of the original sample, the tentative amount of M protein was defined to be the exponential component 3 of the multiplier,
23. Statistical analyses of the data, Inhibitor Library concentration including ANOVA, were carried out using GraphPad Prism version 4.03 (GraphPad software). Differences were considered statistically significant if the P value was <0.05. The DNA fragments of csrRS, including their open reading frame and flanking regions, were amplified through PCR using Pyrobest DNA polymerase. PCR was conducted under the following conditions: 94°C for 5 min, followed by 30 cycles each consisting of 94°C for 30 s, 45°C for 30 s and 72°C for 3 min, and finally 72°C for 7 min. The primers csrR-n3 and csrS-c5 were used for the PCR reaction. The following primers were used for sequencing: csrR-n4; csrR-n6; csrS-n2; csrS-n4; csrS-c4; csrS-c6; csrS-c7 and csrS-c10. The primers mga-c5 and BIBW2992 in vivo mga-n3 were used to amplify the mga gene and the flanking region by means of conventional PCR using Pyrobest DNA polymerase. The following primers were used in the sequence analysis: mga-c5; mga-n3; mga-c1; mga-c4; mga-n1 and mga-n2. Each PCR product was purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Acquisition of the sequence data was entrusted to Takara Bio. The primers for the sequencing are listed in Table 1. Streptococcus pyogenes was grown in 5 mL of BHIY broth for approximately 18 hr. 4.7 mL of fresh BHIY was then added to 0.3 mL of the overnight culture; because the mRNA of
the M protein is generated largely during the early logarithmic phase and then degenerates rapidly, cells in the phase (OD600 = 0.3∼0.4) were allowed to grow for ∼2.5 hr, then mixed Mirabegron with 2 volumes of RNA Protect Bacterial Reagent (Qiagen) and kept at room temperature for 5 min. Total RNA was subsequently extracted using the RNeasy Protect Bacterial Mini Kit (Qiagen) according to the manufacturer’s protocol. Oligonucleotide primers and probes specific for emm and proS genes were prepared according to a previously described method (17). RT-PCR was performed using the TaqMan One-Step RT-PCR Master Mix Reagents Kit (Applied Biosystems, Foster City, CA, USA). The RT-PCR mixture (50 μl) contained 25 μl of 2 × Master Mix without uracil N glycosylase, 1.