18 research outputs found
Highly Sensitive SERS Detection of Hg<sup>2+</sup> Ions in Aqueous Media Using Gold Nanoparticles/Graphene Heterojunctions
Gold nanoparticles (AuNPs)/reduced
graphene oxide (rGO) heterojunctions were synthesized directly on
SiO<sub>2</sub>/Si substrates via a seed-assisted growth process.
The in situ chemical fabrication strategy has been proven to be quite
simple and efficient for generating highly active surface-enhanced
Raman scattering (SERS) substrates due to synergistic enhanced protocol
from rGO and AuNPs. The SERS substrates with AuNPs/rGO heterojunctions
have been utilized for trace analysis of mercuryÂ(II) ions via thymine–Hg<sup>2+</sup>–thymine coordination. Thereby, very low limits of
detection, i.e., 0.1 nM or 20 ppt for Hg<sup>2+</sup>, can be achieved
in contrast with some other SERS subsrtates, which suggests that the
heterojunctions are appropriate as versatile surface-enhanced substrates
applied in chemical sensing or biosensing
The 3′ UTR of AP-2α contains a MRE for miR-200b/200c/429 family.
<p>A) Diagram of luciferase reporter constructs. The predicted MRE, wild-type or MRE-deleted AP-2α 3′ UTRs were inserted downstream of the firefly luciferase gene of the pmirGLO vector. B) Repression of firefly luciferase by the interaction between miRNA and the predicted MRE. Each luciferase construct was co-transfected with miR-200b, miR-200c or miR-429 mimics into HEK293 cells. At 24 h post-transfection, the luciferase activity was examined. The firefly luciferase activity was normalized to Renilla luciferase activity. The firefly luciferase activity of the cells that were transfected with miRNA mimics was represented as the percent activity relative to that of the cells that were transfected with negative control miRNA mimics. Data are shown as the mean±SD of three independent experiments. **, p<0.01.</p
SNP rs1045385 A>C variation in the AP-2α 3′ UTR inhibits the binding of the miR-200b/200c/429 family and enhances AP-2α expression.
<p>A) Diagram of the binding between miR-200b/200c/429 seed sequence and the AP-2α 3′ UTR with the A or C allele. B) SNP rs1045385 A>C variation in the AP-2α 3′ UTR inhibited the binding of the miR-200b/200c/429 family. HEK293 cells were transfected with indicated miRNA mimics and luciferase reporter constructs containing the AP-2α 3′ UTR with the A or C allele. At 24 h post-transfection, luciferase activity was examined. The firefly luciferase activity was normalized to the Renilla luciferase activity. The firefly luciferase activity of the cells that were transfected with miRNA mimics was represented as the percent activity relative to that of the cells that were transfected with negative control miRNA mimics. **, p<0.01. C) SNP rs1045385 A>C variation in the AP-2α 3′ UTR enhanced the AP-2α expression in HEC-1A cells. The Myc-tagged expression construct of full-length AP-2α with the A or C allele was co-transfected with miRNA mimics into HEC-1A cells. At 36 h after transfection, cell lysates were prepared and subjected to Western blot analysis.</p
Generation of KCTD10<sup>−/−</sup> mice.
<p>(A), the genomic DNA of <i>Kctd10</i> gene includes 7 exons and 6 introns. The genetic manipulation of <i>Kctd10</i> was designed to delete exon 2, the first loxP was inserted into intron 2, and the second loxP together with neomycin-resistant gene flanked by FRT sites was inserted into intron 3. (B–D), Gene disruption was confirmed using Southern blotting in positive ES cells. (E), Genotyping strategy of the mutant mouse, there is only a 670 bp band in KCTD10<sup>−/−</sup> mice, two bands (670 bp and 306 bp) in the KCTD10<sup>+/−</sup> mice, and only a 306 bp in the wild type mice. (F–G), RT-PCR and western blotting results indicate that the deletion is successful. (F), embryos with the same genotype were collected, and total RNA was extracted separately, reverse transcripted into cDNA and real-time PCR was performed to determine the KCTD10 mRNA levels. (G), embryos with the same genotype were collected, and total protein was extracted separately and immunoblotted by anti-KCTD10 antibody.</p
KCTD10 may mediate Notch1 proteolytic degradation by Cullin3.
<p>(A), total proteins were extracted from KCTD10<sup>−/−</sup> embryos and in wild type embryos, endogenous immunoprecipitation was performed to show KCTD10 and cullin3 exist in the immune complex of Notch1 simultaneously. However, rabbit preimmune IgG did not recognize any target protein. (B), HUVECs were transfected with HA-cullin3 and harvested 24 h after transfection, the cell lysate were immunoprecipitated by the antibodis against Notch1, NICD, HA, and KCTD10. The precipitated protein were separated by 10% SDS-PAGE and immunblotted with anti-HA and anti-KCTD10 antibody. (C, D), HUVECs were transfected with an increasing amount of pCMV-HA-KCTD10 (0, 0.5, 1, 2 µg) and treated with MG132 (D), and harvested 8 h after transfection. Cell lysates were prepared and separated using 10% SDS-PAGE electrophoresis. A single blot membrane was cut into strips based on molecular weight and incubated with anti-KCTD10, anti-Notch1 (Santa), and β-actin antibodies to detect the KCTD10, Notch1 and β-actin protein levels, respectively. (E), HUVECs were transfected with an increasing amount of pCMV-HA-KCTD10 (0, 0.5, 1, 2 µg), the total RNA were extracted and the mRNA levels of KCTD10 and Notch1 were detected. (F), HUVECs were treated by increasing amount of DAPT for 2 h, and the KCTD10 and β-actin protein levels were detected using Western blotting.</p
Oligonucleotide primers used in this study and estimated size of PCR products.
<p>The oligonucleotide sequences used in this study are listed in the upper table. The oligonucleotide primers were used to genotype the mice, to obtain genomic fragments of <i>Kctd10</i>. The estimated sizes of PCR products obtained during genotyping the mice are indicated in the lower table.</p><p>Oligonucleotide primers used in this study and estimated size of PCR products.</p
KCTD10 is induced by VEGF in a dose- and time-dependent manner.
<p>(A), HUVECs were incubated with 10 ng/mL VEGF for 0 min, 30 min, 45 min, 60 min, 90 min and 120 min, the cell extractions were subjected to SDS-PAGE, and immunoblotted with polyclonal antibody against KCTD10 and β-actin separately. (B), HUVECs were incubated with 0, 5, 10 ng/mL VEGF for 120 min, the cell extractions were subjected to SDS-PAGE, and immune-blotted with polyclonal antibody against KCTD10 and β-actin separately. (C), HUVECs were incubated with 10 ng/mL VEGF for 0 min, 30 min, and 60 min, and the cells were fixed and immune-stained polyclonal antibody against KCTD10. (D), HUVECs were treated with 10 ng/mL VEGF for indicated time, and the total RNA of the cells were extracted, RT-PCR analysis showing the mRNA levels of KCTD10.</p
KCTD10-deficient embryos show heart defects.
<p>(A), embryos with almost the same somites were cut longitudinally in paraffin, and stained using Hemotoxylin/eosin. The red arrows show the enlarged pericardial edema in mutant embryos. (B), embryos with almost the same somites were cut athwartships in paraffin, and the red arrowheads show the atrioventricular valve defects in the KCTD10<sup>−/−</sup> embryos.</p
statistical of abnormal embryos at E9.5–E14.5.
<p>statistical of abnormal embryos at E9.5–E14.5.</p
Angiogenesis defects is the direct cause for embryonic lethality in KCTD10<sup>−/−</sup> embryos.
<p>(A), the wild type (+/+) and mutant (−/−) embryos at E10.5 were stained using PECAM-1. (B), HE staining of E10.5 embryos. The red arrow indicates the abnormal dorsal aorta in the KCTD10<sup>−/−</sup> embryos. (C), wild type (+/+) and mutant (−/−) embryos at E8.5 and E10.5 were stained using PECAM-1, showing the changes of dorsal aorta (DA) development. (D), the yolk sacs at E10.5 were isolated and fixed, frozen sectioned, and immunostained using PECAM-1. The yolk sac showed disorganized in the KCTD10<sup>−/−</sup> embryos.(E), yolk sacs were dissected from the wild type (+/+) and mutant (−/−) embryos at E10.5 and paraffin sectioned, and then stained using Hemotoxylin/eosin. Enlarged lacunae between the endodermal and mesodermal layers in the mutant (−/−) embryos were observed.</p