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B cell-specific Cks1 overexpression results in elevated Cks1 protein levels.

By Susanne Kratzat (163428), Viktoriya Nikolova (163437), Cornelius Miething (163446), Alexander Hoellein (163454), Stephanie Schoeffmann (163461), Oliver Gorka (163470), Elke Pietschmann (163476), Anna-Lena Illert (163483), Jürgen Ruland (49306), Christian Peschel (163491), Jonas Nilsson (163498), Justus Duyster (163507) and Ulrich Keller (163515)

Abstract

<p><b>A</b>, Immunoblot analysis of Cre-mediated conditional expression of Cks1 in retrovirally infected NIH-3T3 shows the tightness of the translational stop-cassette. Retroviral infection was performed 3 times in 12 hour intervals with 8 µg/ml polybrene. <b>B</b>, Depiction of the murine bone marrow retroviral transduction-transplantation model. 5FU-stimulated bone marrow cells from <i>CD19-Cre</i> transgenic mice were infected 4 times with retrovirus and then intravenously injected into lethally irradiated wild type (wt) recipient mice. <b>C</b>, Flow cytometric analysis of engraftment of GFP-positive B cells in the peripheral blood 28 days after bone marrow transplantation. Left panel: Histogram upon B220 staining. Right panel: Results of two different transplantations are combined. The bars represent the mean ± standard error (n = 7 mice for <i>Stop-GFP</i>, n = 9 for <i>Stop-Cks1-GFP</i>). One experiment was performed with 2×10<sup>6</sup> transplanted cells and an infection rate of 15% (GFP positivity, n = 4 or 5 recipient mice), and one with 1×10<sup>6</sup> cells and an infection rate of 45% (GFP positivity, n = 2 or 3 recipient mice). <b>D</b>, realtime PCR analysis of <i>Cks1</i> transcript levels in the indicated cells. <i>Stop-GFP</i> (n = 2), <i>Stop-Cks1-GFP</i> (n = 3). The bars represent the mean ± standard deviation. <b>E</b>, Immunoblot anaylysis of Cks1 protein levels in B220+GFP+ B cells. The B cells were FACS-sorted from n = 6 mice of each of the indicated treatment groups 50 days after transplantation.</p

Topics: Cell Biology, Cancer, Hematology, cell-specific, cks1, overexpression, elevated
Year: 2012
DOI identifier: 10.1371/journal.pone.0037433.g002
OAI identifier: oai:figshare.com:article/306957
Provided by: FigShare
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