FIG 2.

O. tsutsugamushi Anks interact with GST-SKP1. (A) GST-SKP1 coprecipitates FLAG-tagged Anks. Lysates of transfected HeLa cells expressing GST-SKP1 together with FLAG-tagged Anks, positive-control FLAG-SKP2, or negative control FLAG-BAP were incubated with glutathione-Sepharose to precipitate GST-SKP1 and thereby coprecipitate interacting proteins. The resulting Western blots were screened with FLAG antibody. (B) GST-SKP1 coprecipitates GFP-tagged Anks. Lysates of transfected HeLa cells expressing GST-SKP1 together with GFP-tagged Ank9, Ank10, Ank14, or negative-control GFP alone were Western blotted and screened with GFP antibody. (C) FLAG-Ank9 coimmunoprecipitates GST-SKP1. Lysates of transfected HeLa cells expressing GST-SKP1 together with FLAG-Ank9 or FLAG-BAP were incubated with FLAG antibody-conjugated agarose beads to immunoprecipitate FLAG-tagged proteins and coprecipitate interacting proteins. The resulting Western blots (WB) were probed with GST antibody (α-GST). Precipitation of FLAG-tagged proteins was confirmed by probing the stripped blots with FLAG antibody (α-FLAG). (A to C) For each experiment, expression of the protein of interest was confirmed in the “Input” lane containing 3% of the sample. The pulldown (PD) lanes represent proteins that coprecipitated with GST-SKP1 (A and B) or coimmunoprecipitated with FLAG-tagged protein (C). Precipitation of GST-SKP1 was confirmed, but is not shown, for each sample in panels A and B. The arrows indicate the expected sizes of the prey proteins. All the blots are aligned at the 50-kDa marker, as indicated on the leftmost blot in each row. The data presented are representative of two to five experiments with similar results.