Figure 5

Assessing the importance of cellular BSG for extracellular PPIA activity on monocytes. A) Top: ERK1/2 phosphorylation was monitored in MOLM13 cells using a flow cytometric assay (as in Figure 4) for both sh0 cells (solid line) and shBSG cells (dashed line). Bottom: Western blot analysis using the BSG antibody shows that shBSG cells exhibit a significant knockdown when compared to sh0 cells. No significant increase in cellular BSG expression was observed in sh0 cells within this 30 min timeframe as assessed by western blot analysis. B) Top: In MOLM13 cells, IL-8 luciferase reporter activity was monitored after stimulation with recombinant PPIA. Bottom: Cellular BSG was monitored via western blot analysis both before and after 24 h stimulation with recombinant PPIA. C) In U937 cells, a similar analysis was conducted as in (B). For all stimulations, 25 μM recombinant PPIA was used and β-actin was used as a loading control. Arrows denote both highly and lowly glycosylated BSG as well as the β-actin loading control.