Determination of the Ch-7TM protein expressed in the adherent cells/1 h and 48 h of chicken PBMC.

<p>MAbs B28D5 and KUL01 were used in immunofluorescent double staining (A and B) and western blotting (C). Cells were grown on coverslips and fixed in cold acetone for 20 min. The adherent cells/1 h (A) and adherent cells/48 h (B) were visualized with PE-conjugated KUL01 antibody (right top), and subsequently double-stained with the Ch-7TM protein, using MAb B28D5 and Alexa 488-conjugated rabbit anti-mouse IgG as the primary and secondary antibodies, respectively (right bottom). DAPI was used for nuclear counterstaining (left bottom), and visualization was conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086880#pone-0086880-g003" target="_blank">Fig. 3C</a>. Bar: 10 µm. (C) Western blot analysis of the Ch-7TM protein. The PBMC was cultured for 1 h; the adherent cells were scraped and used as the adherent cells/1 h. Some of the adherent cells were cultured for an additional 48 h and treated as the adherent cells/48 h. The adherent cells/48 h (Lane 1) and adherent cells/1 h (Lane 2) were prepared for SDS-PAGE, and the proteins were transferred onto the PVDF membrane. The 1∶1×10⁴ dilution of B28D5 and 1∶5×10⁵ dilution of HRP-coupled goat anti-mouse Ig (Thermo Scientific, Pierce Biotechnology) were used as the primary and the secondary antibodies, respectively. The signals were enhanced using the SuperSignal® West Femto Maximum Sensitivity Substrate kit (Thermo Scientific). Molecular weights of the prestained standard markers (Fermentas) are indicated on the left.