Fig. 9.

Identification of an A. phagocytophilum Rab10-specific ligand.

A. GST-Rab10 beads pull down A. phagocytophilum UMPK (APH0111). Glutathione sepharose beads coupled with GST-Rab10 or GST alone were incubated with whole-cell lysates of uninfected or A. phagocytophilum-infected HL-60 cells. Eluates from pulldown beads and beads alone were resolved by SDS-PAGE and stained with Sypro Ruby. The band of approximately 26 kDa (denoted by asterisk) that was unique to the eluate from GST-Rab10 beads incubated with A. phagocytophilum-infected cell lysate was excised and analysed by mass spectroscopy. PD, pulldown. U, uninfected. I, infected.

B. A. phagocytophilum UMPK amino acid sequence. Underlined peptide sequences are those that were identified by LC-MS/MS following precipitation of UMPK by GST-Rab10. Peptides that were identified as being on the A. phagocytophilum surface in our prior report (Kahlon et al., 2013) are denoted by green highlighting. Blue highlighting demarcates predicted transmembrane domains. Red highlighting indicates the peptide encompassing amino acids 182–195 against which antiserum was raised.