Fig. 1

ERRα directly interacts with p53. a FreeStyle^TM 293-F cells transiently transfected with the pcDNA3 empty vector (mock) or pCMV flag ERRα were subjected to an affinity pull-down assay by using an ANTI-FLAG®M2 Affinity Gel. Proteins were stained with crystal violet (E1-E4, eluted fractions; B, beads). b After excision of the upper and lower bands from the gel, both samples were purified and further examined by LC-MS/MS. Overall, two independent experiments were performed in triplicate to identify ERRα (upper band) and p53 (lower band). c 293T cells were transiently transfected with the pcDNA3 empty vector (mock), pCMV flag ERRα, and pcDNA3 flag p53. Cells were then subjected to an affinity pull-down assay as for a and immunoblot (IB) analysis with monoclonal Anti-Flag®M2, anti-ERRα (right), anti-p53 (left), and anti-GAPDH as loading control for both. d Endogenous immunoprecipitation (IP) of ERRα (left) and p53 (right) was performed by using 293T cells followed by IB analysis with anti-ERRα (right), anti-p53 (left), and anti-GAPDH. e HCT-116^p53+/+ (right) and HCT-116^p53−/− (left) cells were used to perform endogenous IP followed by IB analysis with anti-ERRα, anti-p53 (two different antibodies), and anti-GAPDH. f HCT-116^p53+/+ (top) and HCT-116^p53−/− (bottom) cells were used to perform a proximity ligation assay followed by analysis by fluorescence microscopy. Scale bar, 50 μm. g HCT-15 (left) and DLD-1 (right) colon cancer cell lines were used to perform endogenous IP followed by IB analysis with anti-ERRα, anti-p53, and anti-GAPDH. h Modeling of the ERRα LBD interacting with the p53 CTD (ERRα LBD, yellow; p53 CTD, blue). See additional file 2: Figure S2 for more details. i 293T cells were transiently transfected with the pcDNA3 empty vector (mock), the pcDNA 3.1 ERRα LBDΔ190-423 with V5 Epitope/His tag, and the pcDNA3 p53 CTDΔ300-393 with a Flag tag. Cells were then subjected to an affinity pull-down assay as for a. This was followed by IB analysis with monoclonal Anti-Flag®M2 and the rabbit monoclonal anti-ERRα, which recognizes the endogenous level of the protein in the residues near the carboxy-terminus of the human ERRα protein. Data represent protein-protein interaction and were not quantified