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Thanks a lot. I did a PubMed search for "HeLa cells and ARF", and found a paper, which shows the band detected by the same antibody as that I obtained from your company. I am looking for some HeLa cells, hopefully to get some positive control samples. Have a nice weekend. Oncogene. 2002 Oct 3;21(44):6779-90. Human p14(Arf): an exquisite sensor of morphological changes and of short-lived perturbations in cell cycle and in nucleolar function. David-Pfeuty T, Nouvian-Dooghe Y. UMR 146 du CNRS, Institut Curie-Recherche, Batiment 110, Centre Universitaire, 91405 Orsay Cedex, France. Therese.Pfeuty@curie.u-psud.fr The human Ink4a/Arf tumor suppressor locus encodes two distinct products: p16(Ink4a) which prevents phosphorylation and inactivation of the retinoblastoma protein and, p14(Arf), a nucleolar protein which activates the function of the tumor suppressor p53 protein in the nucleoplasm in response to oncogenic stimulation through an as yet ill-defined mechanism. Here we show that the level of endogenous p14(Arf) and its balance between the nucleolus and the nucleoplasm in HeLa cells are exquisitely sensitive to changes in cell morphology and to short-lived perturbations in cell cycle and in nucleolar function such as those induced by the cyclin-dependent kinase inhibitor, roscovitine, and the casein kinase II and RNA synthesis inhibitor, DRB. Most remarkably, whereas p14(Arf) predominantly concentrates in the nucleolus of interphase cells and transiently disappears between metaphase and early G1 under normal growth conditions, it massively and reversibly accumulates in the nucleoplasm of postmitotic and S-phase cells upon short-term treatment with roscovitine and, at a lesser extent, DRB. In line with the fact that the nuclear level of p53 reaches a peak between mid-G1 and the G1/S border in p53-expressor cells which lack Arf expression, these results provide a clue that, in p53+/Arf+ cells, Arf proteins might serve both to speed and to amplify p53-mediated responses in conditions and cell cycle periods in which the mechanisms involved in p53 stabilization and activation are not fully operational. They further suggest that human endogenous p14(Arf) might activate p53 pathways in physiologic situations by acting inside the nucleoplasm, especially when normal cell cycle progression and nucleolar function are compromised.
Asked on Jun 25 2004