Fig. 2
The nuclear APP-CT50 complex associates to p53 and PML. (A) FE65 (red, mCherry) and TIP60 (blue, BFP), which are known to co-localize and form a dot-like structure, were transfected into HEK293T cells. With the addition of the tumour suppressor protein p53 (green, EGFP), the same pattern could be revealed (for example see white arrows), proving further co-localization and interactions of the proteins. (B) Co-localization of the protein complex is proven by tracking of the fluorescence intensity along the indicated arrow, which revealed peak intensities of each component (green: p53-EGFP, red: FE65-mcherry, blue: TIP60-BFP) supporting the association of all components within one complex. (C) Omitting FE65 and transfecting only p53 and TIP60 also revealed a co-localization (for example see white arrows), proving that the complex is independent of FE65. (D) P53 interaction with the APP-CT50 complex (FE65-EGFP/ TIP60-HA co-transfected to p53-EGFP versus control (EGFP-NLS, nuclear localization sequence)) was validated by co-immunoprecipitation (left side: input blot, right side: elution blot). IP using anti-HA tag antibody (against TIP60-HA) revealed precipitation of FE65 (white arrow) as well as of p53 (red arrow). Respective controls did not show a co-precipitation. TIP60-HA precipitation also occurred using anti-GFP as bait in well agreement to results obtained in part C (white arrowhead). Notably, high levels of endogenous p53 co-eluted in the same condition (red arrowhead), whereas a moderate endogenous p53 signal was observable in control conditions (red arrow). (E) A second tumour suppressor protein was identified to associate with the APP-CT50 complex: the promyelocytic leukemia protein PML. Different phenotypes of association were observed, e.g. one or two APP-CT50 (blue, BFP)/FE65 (red, mCherry) dots (TIP60-HA was co-transfected w/o fluorophore) associated with a single PML (green, EGFP) aggregate (first and second zoom-in row, compare fluorescence intensities). Alternatively, large APP-CT50/FE65 complexes with enclosed PML-dots were found (third row)