FIG 6.

GST-SKP1 precipitates O. tsutsugamushi-derived Ank9. HeLa cells expressing GST-SKP1 were infected with O. tsutsugamushi for 48 h prior to lysis and GST pulldown analysis. Uninfected HeLa cells served as a negative control. (A) Ank9 antiserum recognizes a host cell background protein with an apparent molecular mass similar to that of Ank9. To confirm infection, lysates (Input) were subjected to Western blotting with O. tsutsugamushi OmpA antibody. Probing the lysates with Ank9 antiserum detected a background band in both uninfected and infected cell lysates that was the same size as that expected for Ank9 (∼47 kDa). (B) Ank9 antiserum detects O. tsutsugamushi-derived Ank9 coprecipitated by GST-SKP1. To verify that O. tsutsugamushi expresses Ank9 during infection and that it is capable of interacting with SKP1, Western blots of proteins coprecipitated by GST affinity resin were probed with Ank9 antiserum. Screening with GST antibody confirmed expression and precipitation of GST-SKP1. Protein sizes are indicated on the left. The arrowheads point to bands of interest in each blot. The data are representative of at least two experiments with similar results.