FIG 5.

Ectopically expressed Ank4 precipitates endogenous Bat3 but does not alter Bat3 cellular levels. (A) Flag-Ank4 precipitates Bat3 in an F-box-independent manner. Whole-cell lysates of transfected HeLa cells expressing Flag-tagged BAP, Ank4, or Ank4ΔF-box were incubated with Flag affinity resin to precipitate Flag-tagged proteins and coprecipitate interacting proteins. Eluted proteins were analyzed by Western blotting and screened with Bat3 antibody. Input samples corresponding to 3% of each lysate used for precipitation were also probed. Vertical lines between the lanes in the input samples indicate that irrelevant lanes of the blot were removed. Flag protein expression and pulldown were confirmed by stripping and reprobing the blots with Flag antibody. (B and C) Flag-Ank4 does not alter endogenous Bat3 cellular levels. Whole-cell lysates of HeLa cells transfected to express Flag-BAP, Flag-Ank4, or Flag-Ank4ΔF-box were subjected to Western blotting using Bat3, Flag tag, and GADPH antibodies (B). The means ± standard deviations of the ratios of Bat3 to GADPH densitometric signals from three separate experiments were determined (C). Data are representative of three experiments with similar results.