Fig. 5
Mutant p53 associates with MCM2, MCM4 and MCM7 on chromatin. a Protein levels of MCM2, MCM4, MCM7, mtp53 and fibrillarin in the chromatin fraction were determined by Western blot analysis in MDA-468.shp53 cells grown in the presence or absence of 8 μg/ml of doxycycline for 12 days, and HT-29 colon cancer cells transfected with p53-siRNA (p53) or control siRNA (Con). b Analysis of p53/MCM2 complexes (red) by immunofluorescence microscopy in combination with in situ proximity ligation assay (PLA) in MDA-468 vector and MDA-468.shp53 cells grown in the presence of 8 μg/ml of doxycycline for 12 days. DNA was counterstained with DAPI (blue) and GFP (green) was an indicator of doxycyline-mediated induction. The z stack confocal maximum intensity projection images of p53/MCM2 and DAPI, p53/MCM2 and GFP are shown. c Co-immunoprecipitation (co-IP) of MCM2 and MCM4 with exogenously expressed mtp53 (R175H) and wtp53 in H1299 cells. Whole cell lystates of H1299 cells transfected with wtp53 or mtp53 were incubated with anti-p53 antibody followed by immunoblot with anti-MCM2 or anti-MCM4 antibodies. d Co-IP of MCM4 and mtp53 in thymic lymphomas from mtp53 (Trp53R172H/R172H) mice. Thymic lymphomas from mtp53 mice and p53−/− mice as well as normal thymic tissue from mtp53 mice were subjected to co-IP assays using anti-p53 antibody followed by immunoblot with anti-MCM4 antibody
