Precise Measurement of B Meson Lifetimes with Hadronic Decay Final States

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特集9 : 研究解説 : 射出成形における温度計測技術

射出成形機の加熱シリンダ内における溶融樹脂温度計測手法として赤外線放射温度計測法，シース熱電対を応用した方法，また金型内樹脂の温度計測手法として超音波計測法，熱電対法，蛍光法を取り上げ，原理と事例，問題点について解説を行い，併せて著者らの開発による集積熱電対センサについて，前記手法と比較しながらその可能性を紹介した．特集 生産加工システムの先進技術departmental bulletin pape

Time course of changes in cell cycle related proteins level after serum withdrawal and serum readdition to WS1 cells.

<p>The proteins were determined in an immunoblot with specific antibodies for each protein, VRK1, p27, PCNA and phospho-Rb. The level of actin was used as a loading control. At the bottom is shown the quantification of the level of each protein. In the case of VRK1 it is also shown the quantification by RT-PCR of its RNA (dashed line) at the same time points. These experiments were independently performed three times.</p

VRK1 levels in different growth conditions of human fibroblasts.

<p>(A). Flow cytometry profile of WS1 cell cycle in non-synchronized (left) and serum starved cells (right). (B). Nuclear localization of VRK1 and morphology of WS1 cells in non-synchronized and starved WS1 cells. Endogenous VRK1 protein was detected with a specific polyclonal antibody (VE1) in the cell nuclei. The size bar represents 50 µm. (C). Level of VRK1 endogenous protein determined in an immunoblot. (D). Change in VRK1 gene expression by qRT-PCR upon withdrawal or addition of serum to the cell culture. These experiments were independently performed three times.</p

Stability of endogenous or transfected VRK1 protein.

<p>(A) The variation in levels of endogenous VRK1 and p53 proteins in HeLa cells was followed at different time points after addition of cycloheximide. The p53 protein drops in less than 30 minutes as expected, but there is no detectable change in VRK1 protein even after twelve hours suggesting it has a very high stability. (B). HEK293T cells were transfected with plasmids expressing wild-type human VRK1 or its kinase-dead K179E mutant. The stability of both proteins was followed at different time points after cycloheximide addition. Cells were transfected with plasmids pCEFL-HA-VRK1 or pCEFL-HA-VRK1(K179E) as previously described (Vega et al., 2004). These experiments were independently performed three times.</p

Effect of siRNA on endogenous VRK1 and cell proliferation.

<p>(A). Proliferation of WS1 cells that were transfected with siRNA control or siRNA specific for VRK1. The total cell number in the dish was determined at different time points (top). The endogenous protein was detected in an immunoblot (bottom) (B) Photograph of the WS1 cell cultures treated with siRNA control (top) or siVRK1 (bottom). The proliferation is much slower in cells transfected with the specific siVRK1. These experiments were independently performed three times. (C). Effect on VRK1 RNA levels in WS1 cells transfected with either siControl or siVRK1. NTC: non transfected cells.</p

Effect of siVRK1 on proteins related with cell cycle progression.

<p>Levels of proteins in non-transfected, transfected with siRNA control, or with siRNA specific for VRK1 were determined in after 120 hours post transfection. The proteins were determined with the corresponding antibody indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001642#s4" target="_blank">methods</a> section, and identified in the corresponding immunoblot. These experiments were independently performed five times.</p

Effect of p53 phosphorylation mutants on the downregulation of VRK1.

<p>The p53 aminoacid substitutions are indicated in the diagram. At the top is shown the level of expression of each p53 mutant and at the bottom is shown the quantification of the blot. As positive control is included wild-type p53 that induces the effect (second lane). As negative control for lack of effect the p53^R280K mutant is also included. The constructs have mutated either individual, or different combinations of residues as indicated in the legend, or alternatively all p53 phosphorylation residues in the N-terminus (substitutions of phosphorylable residues in the N-terminal region; ΔN: S6A, S9A, S15A, T18A, S20A, S33A and S37A), the C-terminus (substitutions of phosphorylable residues in the C-terminal region; ΔC: S315A, S371A, S376A, S378A, S392A) and both (ΔN/ΔC, all phosphorylatable residues in the N and C-terminal region are substituted). H1299 cells were transfected with 5 µg of pCEFL-HA-VRK1 and the indicated p53 construct. The expression of the p53 constructs aimed to express similar protein levels of all mutants. Cell extracts were prepared 36 hours after transfection and the levels of both proteins were determined by western blot. The transfected VRK1 was detected with an antibody specific for the HA epitope. p53 was detected with a mixture of DO1 and Pab1801 antibodies. The experiments were performed independently three times. The quantification corresponding to the immunoblots shown and is presented in lower bar graphs.</p

PCAF does not protect from p53-induced downregulation of VRK1.

<p>(A) PCAF over-expression does not exert any protective effect on VRK1 downregulation by p53. H1299 cells were transfected with pCB6+p53 (0.2 µg) and pCEFL-HA-VRK1 (5 µg), and the experiment was carried out like in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002649#pone-0002649-g001" target="_blank">Figure 1</a>, but a PCAF expression plasmid was included instead of the p300 plasmid. VRK1 protein levels were quantified and shown in the graph. PCAF was detected with an anti-Flag antibody. VRK1 was detected with an anti-HA antibody. The quantification of immunoblots is presented in bar graphs. (B) PCAF over-expression by itself, in the absence of p53, does not have any effect on VRK1 protein levels. Normalized VRK1 protein levels are represented in the graph. The quantification of immunoblots is presented in bar graphs. (C). Increasing amounts of PCAF are unable to compete with p300 in its protection of VRK1 downregulation induced by p53. (D). Increasing amounts of p300 protect from p53-induced downregulation of VRK1 independently of the presence of PCAF. The experiments were performed independently three times.</p

Changes in VRK1 RNA level upon serum withdrawal or readdition to the culture.

<p>(A). Quantification of the VRK1 gene expression in WS1 fibroblasts at different time points upon serum withdrawal (top) and serum readdition to the cell culture (bottom) which was previously serum-deprived for five days. These experiments were independently performed three times. (B). Regulation of the human proximal promoter of human VRK1 by serum. WS1 cells were transfected with plasmid pGL2-B-VRK1(−1028+52). The luciferase activity was determined under different experimental conditions. Control activity is that of cells in the continuous presence of serum. The result is the mean of three experiments.</p