FIGURE 5.

LL-37 binds to the CsrS extracellular domain. A, the CsrS extracellular domain fused to maltose-binding protein (MBP-ECD), the CsrS extracellular domain with three point mutations at acidic residues fused to maltose-binding protein (MBP-ECD 3× mutant), or MBP alone was mixed with LL-37 and then pulled down with amylose resin. Left panel, Coomassie-stained SDS-PAGE of proteins eluted from the amylose resin demonstrating similar amounts of MBP-ECD, MBP-ECD 3× mutant, and MBP alone as a negative control. Right panel, anti-LL-37 Western blot of the amylose eluate. B, MBP-ECD was mixed with LL-37 or with RK-19 and then pulled down with amylose resin. Left panel, Coomassie-stained SDS-PAGE of proteins eluted from the amylose resin demonstrating similar amounts of MBP-ECD. Right panel, Western blot of eluates with anti-LL-37 antibodies that recognize both LL-37 and RK-19. C, MBP-ECD was mixed with biotinylated LL-37 or biotinylated SMAP 29 and then pulled down with amylose resin. Left panel, Coomassie-stained SDS-PAGE of proteins eluted from the amylose resin demonstrating equal loading of MBP-ECD. Right panel, in-gel detection of biotin-labeled LL-37 and SMAP 29 with streptavidin DyLight.