Fig 2: 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stably transduced to overexpress V5-tagged intact p53R273H (FL) or a p53R273H mutant in which residues 331–335 have been replaced by Ala (5A).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.)

Fig 3: EGFR activity regulates binding of DOK2 to EGFR and DOK2 localization.(A) Co-immunoprecipitation of DOK2, EGFR, and RASA1. HEK293 cells were co-transfected with pCMV-DOK2 (all conditions) and either pcDNA3.1 empty vector or pcDNA3.1-EGFRWT, pcDNA3.1-EGFRL858R, or pcDNA3.1-HA-HRASG12V. Cells were serum starved overnight in medium containing 0.1% FBS before stimulation with 50 ng/ml EGF for 2-5 minutes. DOK2 and associated proteins were immunoprecipitated from extracts of stimulated and unstimulated cells with an anti-DOK2 antibody and then analyzed by Western blotting. Upper panels show the proteins in the immunoprecipitation fraction; lower panels show proteins from the total lysate. A black arrowhead indicates HA-HRASG12V whereas a white arrowhead indicates endogenous RAS. Data shown is representative of results from at least three independent experiments. (B) Immunofluorescence of NIH3T3 cells stably expressing WT or EGFRL858R. Cells were transfected with pCMV-DOK2, incubated overnight, serum starved, and then either fixed or stimulated with EGF before fixation. The left panels show DOK2 (green) and DAPI (blue). White arrowheads indicate membrane-associated DOK2 staining. A Western blot indicates total levels of EGFR in these cells (right panel). Data shown is representative from at least three independent experiments. (C) Confocal microscopy analysis of colocalization of DOK2 and EGFR. 3T3-EGFRL858R cells were transfected with FLAG-DOK2 and probed with anti-FLAG (red) or anti-EGFR (green) antibodies. Arrowheads indicate colocalization (yellow) in the panel showing the merged signals. Data shown is representative from at least three independent experiments.

Fig 4: OSS-induced expression of ITGA9 and FOXC2 in HDLEC after EPHB4 or RASA1 knockdown or after expression of EPHB4 2YP(A) Schematic showing design of experiments in (B and C).(B) Western blot analysis of OSS-induced expression of ITGA9 and FOXC2 in HDLEC treated with control, RASA1, or EPHB4 siRNA, or control lentivirus or lentivirus encoding EPHB4 2YP. Blots were probed with anti-GAPDH antibodies to demonstrate equivalent protein loading.(C) Quantitative RT-PCR analysis of FOXC2 mRNA expression in HDLEC transduced with control lentivirus or lentivirus encoding EPHB4 2YP subject to OSS for 48 hours in the presence or absence of AZD6244 (n=3 each condition). Fold increases are relative to HDLEC under static conditions without AZD6244 for each HDLEC type. * P<0.05, ** P<0.01, two-tailed Student’s t-test.