Identification of the Ank1 and Ank6 domains that are essential for the ability to co-immunoprecipitate p65, importin β1, and exportin 1.
Whole cell lysates of HeLa cells expressing the indicated Flag-tagged proteins were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate Flag-tagged proteins and their interacting proteins. (A) Western blots of input lysates and immunoprecipitates were probed with p65, exportin 1 (Exp 1), and importin β1 (Imp β1) antibodies. Expression of each Flag-tagged protein of interest was confirmed by subjecting input lysate to Western blotting using Flag antibody. GAPDH immunoblotting of input lysates served as a loading control. (B) Mean + SD of co-immunoprecipitated p65, exportin 1, or importin β1. Values are presented as the percent change in densitometric signal relative that for each respective protein of interest that was coimmunoprecipitated by Flag-tagged Ank1 or Ank6. Data presented were calculated from three separate experiments formed in (A). Statistically significant (*P < 0.05; **P < 0.01) values are indicated.