5 h. HRP-conjugated donkey anti-goat was used as the secondary antibody and the reaction was developed with a TMB substrate (Tiangen). After 15 min of color development, the stop solution (8.5 M acetic acid, 2.5 M H2SO4) was added and the A450 nm

was recorded. The binding of HDL to the GAS (Type M6 and M41) was tested using GAS cells that were either immobilized onto microplate wells or were in suspension. GAS cell suspensions were added to microplate wells and incubated at room temperature for 1.5 h. Wells were washed and blocked overnight with 200 μL of 1% bovine serum albumin (BSA) in TBS or TBST at 4 °C. HDL binding was performed as described above in the rScl1 binding ELISA. find more For the HDL binding to GAS cells in suspension, cells were incubated for 1.5 h with 1% BSA in TBS or TBST. After washing with TBS or TBST, 100 μL of human plasma was added to 1 mL of the cells. Following a 1-h incubation at room temperature, bacterial cells were pelleted and washed three times with TBS or TBST. After the final centrifugation, cell-bound proteins were dissociated Tofacitinib cell line from the cells by the incubation with 200 μL of 0.1 M glycine-HCl solution, pH 2, for 15 min. Bacterial cells were then removed by centrifugation and the proteins in the supernatant were precipitated with 10% TCA and analyzed by SDS-PAGE and immunoblotting. Electroblotting was carried out at a constant voltage

of 30 V for 1 h to transfer ApoAI and at a constant current of 300 mA for 3 h to transfer ApoB100. The immunodetection of ApoAI was performed with a goat anti-ApoAI antibody, followed by HRP-conjugated donkey Elongation factor 2 kinase anti-goat secondary antibody as described above. The presence of ApoB100 was tested with a goat anti-LDL antibody (Chemicon, CA), followed by HRP-conjugated donkey anti-goat antibody (R&D Systems), and the detection was performed with chemiluminescence reagent (Tiangen). Results were expressed as mean±SD. Statistical significance was calculated

using two-tailed Student’s t-test for comparisons of two groups and Student–Neuman–Keuls for comparison of multiple groups, respectively. It was reported previously that several Scl1 proteins interact with LDL/ApoB100 via globular noncollagenous V regions (Han et al., 2006a). Here, we are testing the hypothesis that the Scl1.41 variant possess binding ability to HDL. Recombinant Scl1 (rScl1) proteins C176, C176V, and C176T were constructed, which are derived from Scl1.41 protein of GAS M41-type strain ATCC12373. PCR-amplified DNA fragments corresponding to a full-length or a partial scl1.41-gene sequence were cloned, expressed, and purified in E. coli BL21 (Table 1; Fig. 1a). rScl1 proteins were immobilized onto Strep-Tactin columns through their C-terminal tags (Strep-tag II) and these affinity columns were used to detect Scl1 ligands in human plasma. Human plasma (0.5 mL) was applied to these columns, including the control column without the rScl1 protein.