Although the p53 tumor suppressor/transcription factor often accumulates in the cytoplasm

Although the p53 tumor suppressor/transcription factor often accumulates in the cytoplasm of healthy cells, limited information is available on the cytoplasmic function of p53. Systems (Promega) according to manufacturer protocols. Protein binding was analyzed by co-immunoprecipitation. Where indicated, the expressed proteins were incubated with GST or GST-coupled proteins (Abnova), followed by precipitation with glutathione-conjugated Sepharose (Amersham). The precipitates were analyzed by Western blotting. Expression constructs and mutagenesis Expression constructs were prepared using pcDNA3, pCMV/myc/mito, pEGFP-C1, and pTRE-Tight vectors. The former two vectors were obtained from Invitrogen, while the latter was from Clontech. These vectors were used for the following purposes: pEGFP-C1, for confocal microscopy and intravasation assays; pTRE-Tight, for the expression of pro-apoptotic Bcl-2 members (Bax and Bak); pCMV/myc/mito, for the expression of ND5 and ND5G13289A; and pcDNA3, for all other purposes. p53R175H, p53K305N, p53K305N/R175H, Bcl-wG94A, and ND5G13289A were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) [43]. Animals Female BALB/cAnNCrj-nu/nu mice (6 wks old) were purchased from Charles River. All animal experiments were performed under approved protocols of our Institutional Animal Care and Use Committee. Intravasation assay H460 cells stably transfected with pEGFP-C1 vectors encoding the indicated genes were subcutaneously injected into the hind legs of mice (107/mouse) to form xenograft tumors. Tumor volumes were calculated as described [44]. After 2 weeks, mice were anesthetized, blood was obtained via cardiac puncture, and 0.1 mL of blood was mixed with 2 mL RBC-lysis buffer (Intron Biotech). Cells were collected by centrifugation (350 < 0.05, which was determined by a Student's test or one-way ANOVA using GraphPad software. SUPPLEMENTARY MATERIAL AND FIGURES Click here to view.(863K, pdf) Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2012M2A2A7010459, 2012R1A2A2A01045978, 2008-0062611). Footnotes CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES 1. Muller PA, Vousden KH, Norman JC. p53 and its mutants in tumor cell migration and invasion. J Cell Biol. 2011;192:209C218. [PMC free article] [PubMed] 2. Riley T, Sontag E, Chen Nr2f1 P, Levine A. Transcriptional control of human p53- regulated genes. Nat Rev Mol Cell Biol. 2008;9:402C412. [PubMed] 3. Moll UM, LaQuaglia M, Bnard J, Riou G. 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