Fig 1: p53R273H interacts with p62 via its CTD. (A) Volcano plot of the top hits (red) in immunoprecipitation and mass spectrometry analysis of p53R273H IPs from CTRL PANC-1 cells, compared to p53KO PANC-1 cells. Top hits were defined as having absolute fold change =1.4 (log2 fold-change [FC] = 0.49), P = 0.05, and unique peptides =2. (B) PANC-1 cell lysates were IP with p53-specific antibodies (Santa Cruz FL-393) or IgG CTRL, followed by WB analysis with antibodies against p62 (Abcam 56416); inputs are shown on the left. (C) PANC-1 cell lysates underwent IP with p62-specific antibodies (Santa Cruz D-3) or IgG CTRL, followed by WB analysis with antibodies against p53 (p53-HRP). (D) PLA between p62 and p53, performed on CTRL and shp53 cells using the Duolink PLA kit (Sigma) with tetramethylrhodamine-5-isothiocyanate (TRITC) as a probe (red). Counterstaining was with DAPI (blue) and phalloidin-FITC (green) to visualize nuclear DNA and F-actin, respectively. Quantification of PLA foci is shown below, ****P < 0.0001 (Student’s t test), based on counting of 40 cells. (Scale bar, 10 µm.) (E) PANC-1 cells stably expressing shRNA directed against the 3'UTR of the endogenous p53 mRNA (3'UTR shp53) were transiently transfected to express wtp53, p53R175H, p53R273H or p53R248W. Cell lysates were subjected to IP with antibodies against p62 (Santa Cruz D-3), followed by WB analysis with antibodies against p53 (p53-HRP) or p62 (Santa Cruz D-3). (F) PANC-1 cells stably expressing shRNA directed against the 3'UTR of the endogenous p53 mRNA (3'UTR shp53) were stably transduced to overexpress V5-tagged intact p53R273H (FL) or a p53R273H mutant in which residues 331–335 have been replaced by Ala (5A). Cell lysates underwent IP with antibodies against p62 (Santa Cruz D-3), followed by WB analysis with antibodies against p62 (Santa Cruz D-3) or p53 (anti rabbit V5 CST #13202). RASGAP was used as a loading CTRL. (G) PANC-1 cells as in F were subjected to PLA with antibodies against the V5 tag (reactive with the V5-tagged overexpressed mutp53) and p62. Nuclei were visualized by DAPI staining. (Scale bar, 10 µm.)