Demonstration of elevated amounts of PIM1 and HMGA1 proteins in PECs after adherence by <i>T. vaginalis</i> (<i>Tv</i>).

<p>In this experiment, trichomonads were added to T25 confluent monolayers of PECs (lane labeled+) using a parasite to PEC ratio of 10∶1. PECs without added organisms are labeled with a minus sign (−). This ratio of 10∶1 was chosen because it has been shown to yield at least one parasite attached per epithelial cell in adherence assays and to optimally signal epithelial cells for up-regulation of expression of genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Kucknoor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Garcia1" target="_blank">[27]</a>. After visible attachment to PECs, non-adherent organisms were decanted and fresh PEC medium added followed by incubation at 37°C for an additional 30 min. The flask was then placed directly in ice for detachment of organisms, after which PECs were washed and removed from the flask for preparation of total proteins for immunoblotting using established protocols, polyclonal rabbit antibodies produced in our laboratories, and equal loading of protein onto gels, as detailed previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Under conditions of exposure of PECs with or without <i>T. vaginalis</i>, no change in the amount of other cellular proteins was detected, as evidenced by no changes in the amounts of Akt and Bad, and this served to show equal amounts of total proteins loaded onto gels for SDS-PAGE and immunoblotting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Prebleed rabbit serum was used as the negative control and gave no reactivity.</p