Fig. 2. ANKRD11 interacts with p53 in vivo and in vitro via the ANK domain. (A) ANKRD11 coimmunoprecipitates with endogenous p53. Protein complexes from either MCF-7 (retroviral-mediated GFP-ANKRD11 expression; mitomycin C treated for 6 hours) or HEK293T cell lysates (endogenous ANKRD11) were immunoprecipitated using an anti-ANKRD11 antibody or pre-immune serum. Immunoprecipitated protein complexes were analyzed by western blot analysis using the indicated antibodies. WB, western blot; IP, immunoprecipitation. (B) p53 interacts with the N-terminal region of ANKRD11. HEK293T cells were transiently transfected with constructs expressing one of four different Flag-tagged regions of the ANKRD11 protein or empty pCMV-Tag2 vector as indicated. Protein complexes from total cell lysates were immunoprecipitated using anti-Flag antibody coated Sepharose beads. Inputs (lanes 1-5) or immunoprecipitates (lanes 6-10) were detected by western blot analysis. Low levels of non-specific p53 binding to the Sepharose beads were also detected (lanes 7-10). (C) ANKRD11 interacts with endogenous p53 through the ankyrin repeat domain. HCT116 cells treated with mitomycin C (6 hours) or HEK293T cells were transiently transfected with constructs encoding myc- or Flag-tagged ANKRD11^144-313aa protein (ANK domain) or empty vector, respectively. Protein complexes were immunoprecipitated using the appropriate antibodies and western blotted. (D) ANKRD11 directly interacts with p53 through the ankyrin repeat domain. Purified recombinant GST (lanes 9 and 11) or GST-ANKRD11^144-313aa (lanes 10 and 12) fusion proteins were incubated with either MBP (lanes 9 and 10) or MBP-p53 (lanes 11 and 12) amylose beads. GST (lane 7) and GST-ANKRD11^144-313aa (lane 8) inputs and interacting protein complexes (lanes 9-12) were western blotted using an anti-GST antibody. To confirm their intactness, all proteins were resolved by SDS-PAGE and visualized by colloidal Coomassie Blue staining (lanes 1-6).