Journal: Molecular human reproduction

Article Title: Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

doi: 10.1093/molehr/gaw065

Figure Lengend Snippet: Figure 2 Effect of S1P on the expression of endothelial adherens junction proteins in endothelial cells in the presence of FF from controls and patients at risk of OHSS. Densitometric quantification of (A) phospho-vascular endothelial (p-VE)-cadherin, (B) VE-cadherin, (C) N-cadherin and (D) β-catenin. Representative immunoblots are shown in the lower panel. Data are expressed as means ± SEM normalized to β-actin of three independent experiments using 20 control patients and 20 OHSS patients at risk of OHSS (*P < 0.05).

Article Snippet: The blot was preincubated in blocking buffer (5% nonfat milk, 0.05% Tween 20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated overnight in blocking buffer at 4°C with appropriate primary antibodies: β-actin 1/3000 (sc-1616-R), N-cadherin 1/200 (sc-7939), vascular endothelial (VE)-cadherin 1/100 (sc-9989) and β-catenin 1/200 (sc-59737) (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), phospho-VE-cadherin 1/1000 (MAB1002) (Millipore, MA, USA), VEGF 1/1000 (ab46154) (Abcam, Cambridge, USA), KDR (VEGFR-2) 1/1000 (D5B1) (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Chemical research in toxicology

Article Title: Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

doi: 10.1021/acs.chemrestox.2c00172

Figure Lengend Snippet: Figure 7. 3′-terminus produced by hyTDG-lyase cannot be extended by DNA Pol β but is resolved by APE1. In lane 1, A 5′-FAM-labeled 79 base oligonucleotide containing a U in a U:G mispair (2.5 pmol, 0.2 μM). In lane 2, the oligonucleotide was incubated with UDG (2.5 U, 0.84 pmol, 0.07 μM) for 1 h at 37 °C in CutSmart buffer, and subsequently, the abasic site was cleaved by incubating with APE1 (5 U, 0.18 pmol, 0.01 μM) for an additional 30 min. In lane 3, Pol β (6.2 pmol, 0.5 μM) and E. coli ligase (5 U, 4 pmol, 0.32 μM), dCTP (250 pmol, 20 μM), and NAD+ (325 pmol, 26 μM) were added for an additional 1 h to simulate short-patch BER. Lane 4 was otherwise identical to lane 2, but hyTDG-lyase was used instead of APE1 at 37 °C for 30 min. Lane 5 was otherwise identical to lane 3, except hyTDG-lyase (26.9 pmol, 2.15 μM) was used instead of APE1 at 37 °C. In lane 6, we similarly generated an abasic site that was then cleaved by hyTDG-lyase for 30 min at 37 °C. Then APE1, Pol β, dCTP, and E. coli ligase were added and incubated for an additional 1 h. Lane 6 demonstrates that the AP endonuclease activity of APE1 can clean up the PUA-βME 3′-end produced by the hyTDG-lyase. The complementary strand is labeled with a 5′-Cy5 fluorophore (red). The overlap between the full-length 5′-FAM-labeled oligo and its 5′-Cy5 complement is depicted as yellow.

Article Snippet: Human 8-oxoguanine DNA glycosylase (hOGG1, #NBP1-453180.1mg) and DNA polymerase β (Pol β, #NBP1-72434-0.5mg) were purchased from Novus Biologicals.

Techniques: Produced, Labeling, Incubation, Generated, Activity Assay

Journal: Nature

Article Title: Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases

doi: 10.1038/s41586-020-03069-8

Figure Lengend Snippet: EMSA and structural analysis of NSD3 C with 187-bp NCPs. a , EMSA analysis of NSD3 C with native or Nle-labeled187-bp NCPs. The concentrations of added NSD3 C are indicated above the lanes. The results were repeated from at least three independent experiments. For gel source data, see . b , Superimposition of the 1:1 NSD3-NCP complex with an apo NCP (PDB ID: 1AOI). Histone proteins and the wrapped DNA in apo NCP are colored in yellow and blue, respectively. Histone proteins and the nucleosomal DNA in the NSD3-binding NCP are colored in grey and orange, respectively. NSD3 is colored in green. c , Top (left) and side (right) views of the sharpened cryo-EM density map of the 1:2 NCP-NSD3 C -E1181K/T1232A complex (contour at 3σ level). NSD3 is colored in green. NCP DNA and histone proteins are colored in orange and grey, respectively. d , Top (left) and side (right) views of the inactive state of the NSD3 C -NCP complex. 146-bp NCP structure (PDB code 1AOI) was docked into the density of the 187-bp NCP. The cryo-EM density map was colored in grey and contoured at 1.5σ lever. Additional densities that lay on the top of the nucleosome disk were densities of NSD3.

Article Snippet: Non-radioactive in vitro methylation assays were performed as previously described using 2 μg of enzyme and 2 μg of recombinant nucleosomes (EpiCypher, catalog: 16–2004) in the presence of non-radiolabeled SAM.

Techniques: Binding Assay, Cryo-EM Sample Prep