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. 2013 Feb 1;24(3):222–233. doi: 10.1091/mbc.E12-03-0198

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© 2013 Kienzle et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Depletion of PKD induces a delay in mitotic entry. HeLa cells transfected with a control siRNA (siLacZ) or PKD1- and PKD2-specific siRNAs were synchronized at the G1/S border by a double-thymidine treatment and released for 0, 6, 8, 10, 12, and 14 h. (A) Scheme showing the experimental design. (B) Cells were fixed, stained with propidium iodide to visualize G1, S, and G2/M phases, and analyzed by flow cytometry. The percentage of cells in G1, S, and G2/M phases is indicated. Brackets indicate G1, S, and G2/M phases. (C) At 14 h after release, cells were fixed and stained with a pHistone 3 (Ser-10)–specific antibody and DRAQ5 to visualize the nucleus. The percentage of pH3-positive cells was quantified. n, total number of cells analyzed. Shown is a representative experiment (n = 2). (D) Cells were lysed, and phosphorylation of Cdk1/Cdc2 at tyrosine 15 and histone H3 at serine 10 was detected by Western blot analysis using specific antibodies. Detection of tubulin served as a loading control, and efficient knockdown was verified for PKD2. Lysates were also analyzed using a Cdk1/Cdc2–specific antibody to ensure that total protein levels were unchanged. Detection of tubulin verified equal loading. Shown is a representative experiment (n = 3).