Nucleocytoplasmic shuttling of VP19C.

<p>(<b>A</b>) <b>DAPI staining differentiates monkey (COS-7) and murine (3T3) nuclei.</b> (B) Nucleocytoplasmic shuttling of VP19C via a leucine-rich NES: COS-7 cells were transfected with plasmid VP19C-EYFP or VP19C NES-m-EYFP. 24 hours later, transfected cells were subjected to the interspecies heterokaryon assay. Mouse 3T3 cells were identified by their speckled nuclei when stained with DAPI. (C) Nucleocytoplasmic shuttling of VP19C during HSV-1 infection. COS-7 cells were firstly infected with HSV-1 at MOI of 2. At 12 hour after infection, the HSV-1 infected COS-7 cells were mixed with 3T3 cells and subjected to the heterokaryon assay. Mouse 3T3 cells were identified by their speckled nuclei when stained with DAPI. Each image is representative of the vast majority of the cells observed.</p

Heterokaryon assay cell counts.

a<p>COS-7 cells were transiently transfected with the various expression vectors, and 24 h later a heterokaryon assay was carried out with NIH 3T3 cells or COS-7 cells were infected with HSV-1, and 12 h later a heterokaryon assay was carried out with 3T3 cells.</p>b<p>Mouse nuclei were considered positive if they were present in fused heterokaryons of COS-7 cells expressing VP19C-EYFP or mutant proteins and monkey 3T3 cells contained detectable levels of the expressed protein.</p>c<p>Mouse nuclei were considered negative if they were present in fused heterokaryons of COS-7 cells expressing VP19C-EYFP or mutant proteins and monkey 3T3 cells did not contain detectable levels of the expressed protein.</p

Characterization of the NES and the nuclear export mechanism of the VP19C protein.

<p>(A) Schematic diagram of wild-type VP19C and the putative NES fused with EYFP. (B) Subcellular localization of NES-EYFP and VP19C-EYFP. (C) The NES of VP19C mediated the nuclear export of EYFP via CRM1 dependent pathway. COS-7 cells were transiently transfected with plasmids encoding NES-EYFP and Rev-NES-EYFP (positive control). The cells were incubated in the absence or presence of 10 ng/ml LMB 24 h after transfection. (D) The NES of VP19C mediated the nuclear export of EYFP via RanGTP. COS-7 cells were co-transfected with pNES-EYFP with or without pRan-Q69L-ECFP. Both fluorescent images of EYFP and ECFP fusion proteins were presented in pseudocolor, green and red, respectively. Each image is representative of the vast majority of the cells observed.</p

Confocal images of <i>Arabidopsis</i> leaf protoplasts transiently expressing Nag-mCherry concomitantly with AtGRIP_aa711–776-GFP.

<p>(A) Nag-mCherry was present in Golgi stacks in <i>Arabidopsis</i> leaf protoplasts. (B) AtGRIP_aa711–776-GFP showed dispersed fluorescence in <i>Arabidopsis</i> leaf protoplasts. (C) Merged image showing the Nag-mCherry signal pseudo-colored in red and AtGRIP_aa711–776-GFP in green. Bar: 5 µm.</p

Dimerization analysis of full-length AtGRIP, AtGRIP_aa711

<p>_{–<b>788</b>}<b>, AtGRIP_aa711</b>_{–<b>766</b>}<b> and AtGRIP_aa711</b>_{–<b>776</b>}<b> by yeast two-hybrid assays and confocal imaging of </b><b><i>Arabidopsis</i></b><b> leaf protoplasts that were transiently expressing Nag-GFP concomitantly with AtGRIP_aa711</b>_{–<b>788</b>}<b>-mCherry and AtGRIP_aa711</b>_{–<b>766</b>}<b>-mCherry.</b> (A) The interaction among full-length AtGRIP, AtGRIP (AA711–788), AtGRIP (AA711–766) and AtGRIP (AA711–776) was analyzed by using the yeast two-hybrid system. After 7 days on synthetic plates lacking adenine, histidine, leucine and tryptophan at 30°C, only the combinations of pGBKT7-AtGRIP with pGADT7-AtGRIP (1) and pGBKT7-AtGRIP (AA711–788) with pGADT7-AtGRIP (AA711–788) (2) produced colonies. Neither pGBKT7-AtGRIP (AA711–766) with pGADT7-AtGRIP (AA711–766) (3) nor pGBKT7-AtGRIP (AA711–776) with pGADT7-AtGRIP (AA711–776) (4) generated colonies. (B) Colonies in (1) and (2) tested positive in the X-gal assay, demonstrating that these fusion proteins can interact. Colonies in (3) and (4) did not test positive in the X-gal assay. (C), (F) Nag-GFP showed Golgi stacks in the <i>Arabidopsis</i> leaf protoplasts. (D) AtGRIP_aa711–788-mCherry in <i>Arabidopsis</i> leaf protoplasts showed punctuated fluorescence. (G) AtGRIP_aa711–766-mCherry showed disperse fluorescence in <i>Arabidopsis</i> leaf protoplasts. (E) Merged image showing the AtGRIP_aa711–788-mCherry signal pseudo-colored in red and Nag-GFP in green, demonstrating co-localization of AtGRIP_aa711–788 and the Golgi stacks in the cell (arrows). (H) Merged image showing the AtGRIP_aa711–766-mCherry signal as pseudo-colored in red and Nag-GFP in green. Bars: 5 µm.</p

AtGRIP dimerization analysis by western blot and yeast two-hybrid assays.

<p>(A) (1) Standard molecular weight marker. (2) SDS-PAGE of <i>Arabidopsis</i> protein extract. (3) SDS-PAGE of <i>Arabidopsis</i> protein extract treated with 0.25 mM DTSSP. (4) Western blot analysis using the purified anti-AtGRIP antibody showed a 92 kDa band in lane 2. (5) Western blot analysis using the purified anti-AtGRIP antibody showed a band of more than 220 kDa in lane 3 (arrow). (B) The interaction among full-length AtGRIP, AtGRIP (AA1-605) and AtGRIP (AA605–788) was analyzed by using the yeast two-hybrid system. After 7 days on synthetic plates lacking adenine, histidine, leucine and tryptophan at 30°C, only the combinations pGBKT7-AtGRIP with pGADT7-AtGRIP (1), pGBKT7-AtGRIP (AA1-605) with pGADT7-AtGRIP (AA1-605) (2) and pGBKT7-AtGRIP (AA605–788) with pGADT7-AtGRIP (AA605–788) (3) produced colonies. Neither pGBKT7-AtGRIP (AA1-605) with pGADT7-AtGRIP (AA605–788) nor pGBKT7-AtGRIP (AA605–788) with pGADT7- AtGRIP (AA1-605) generated colonies (4, 5). (C) Colonies in (1)–(3) tested positive in the X-gal assay, demonstrating that these fusion proteins can interact. Colonies in (4) and (5) did not test positive in the X-gal assay.</p

Supporting information with additional method description and evidences. from Uncovering the mechanisms of <i>Caenorhabditis elegans</i> ageing from global quantification of underlying landscahpe

The detail description of network construction and parameter choice. The Introduction to path integrals method, and the calculation of entropy production rate and flux integrals. The evidences for the network regulations, the gene expression value of ageing and rejuvenation states in the bistable landscape, and the regulation strengths of the oscillation dynamics

Multiple alignments of the GRIP C-terminus from different organisms, namely <i>Arabidopsis thaliana</i>, <i>Chlamydomonas reinhardtii</i>, <i>Oryza sativa</i>, <i>Physcomitrella patens</i> and <i>Populus trichocarpa</i>.

<p>This image shows that the GRIP C-terminus is conserved in the plant kingdom. The locus of each protein used for NCBI alignment was as follows: <i>Arabidopsis thaliana</i> AED98143, <i>Chlamydomonas reinhardtii</i> EDP02398, <i>Oryza sativa</i> BAC83565, <i>Physcomitrella patens</i> EDQ81377 and <i>Populus trichocarpa</i> XP_002306424.</p

Additional file 1 of Long noncoding RNA and messenger RNA profiling in epicardial adipose tissue of patients with new-onset postoperative atrial fibrillation after coronary artery bypass grafting

Additional file 1: Table S1. Primer lists were used for Quantitative real-time polymerase chain reaction

Table_S2B

Population genomic statistics for NS sites for each bin of KA values for mel