Figure 3.

Ankrd2 interacts and colocalizes with p50. (a) IP of Ankrd2 following transfection of C2C12 cells with FLAG-tagged WT or Ser99Ala mutant Ankrd2. Ankrd2 is able to interact with p50, but not with p65. Moreover, Ankrd2 Ser99Ala does not bind to p50, demonstrating that Ankrd2 phosphorylation is essential for p50 binding. (b) Reverse experiment confirming the interaction between Ankrd2 and p50: IP of p50 following transfection of C2C12 cells with FLAG-tagged p50. (c) Immunostainings of proliferating and differentiating C2C12 cells for Ankrd2 and p50. Both proteins were visible in a speckled pattern in the nuclei of proliferating and early differentiating myoblasts (panel I at two different magnifications and panel II). With progressive differentiation, Ankrd2 became almost completely cytoplasmic and did not colocalize with p50 (panels III and IV at two different magnifications). MB, proliferating myoblasts, MT, differentiating myotubes at 1, 3, and 6 days (d) after induction