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. 2016 Sep 7;6:32626. doi: 10.1038/srep32626

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Copyright © 2016, The Author(s)

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(a) Colocalization of wild-type p53 with EGFP-tagged G13R-actin in the perinucleus. Over-expressed wild-type p53 was stained with the Alexa-546-tagged anti-p53 antibody (upper panels). Wild-type p53 (stained with Alexa-488-tagged anti-p53 antibody) did not show any positive colocalization with F-actin (stained with mCherry-Lifeact) (middle panels). The selected ROIs have been magnified in the inset images where the pixels showing intense colocalization are marked with arrowheads. Image magnification: 60×. Scale bar: 10 μm. Three-dimensional (3D) confocal microscopy revealed punctated colocalized yellow pixels of EGFP-G13R-actin and wild-type p53, shown by arrowheads. 3D-reconstruction image shows no correlation between mCherry Lifeact and wild-type p53. Pearson’s colocalization correlation (r) between red and green channels is presented in colocalization scatter plots (inset images, lower panels). (b) Physical association between wild-type p53 and G-actin, at increasing concentrations (0.1, 0.5, 1.0, 2.0 μg) of G13R-actin-EGFP cDNA, indicated in the upper panel. Association of wild-type p53 and G-actin in control and Dox-treated condition were presented in the lower panel. GAPDH was used as the internal loading control. Data are representative three independent experiments. (c) His pull-down assay shows purified p53(1–393)His and purified monomeric G-actin directly interacts in vitro. The purified His6-p53 protein has been detected in the Ni-NTA column ‘elute’ fraction, upper panel. Purification of G-actin (lower panel, left). Purified His6-p53 (400 μg) was incubated with 170 μg of monomeric G-actin in Ni-NTA column. Coomassie staining reveals the position of p53His and G-actin in ‘bait-prey’ complex (lower panel, right). (d) Post-simulation p53:G-actin dynamic complex, in surface/electrostatics (upper panel), p53 in surface/electrostatics and G-actin in cartoon/secondary structure (middle panel), p53 in cartoon/secondary structure and G-actin in surface/electrostatics (lower panel). p53 and G-actin have been shown in cyan and red color respectively.