PMC:6145447 / 19194-21000
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/6145447","sourcedb":"PMC","sourceid":"6145447","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6145447","text":"Chromatin binding assay and western blot\nPreparation of Low-Speed Supernatant (LSS) Xenopus egg extracts and isolation of demembranated sperm nuclei (chromatin) were performed as previously described 34. For chromatin binding experiments, 15 μl of LSS extracts were supplemented with demembranated sperm nuclei (2,500 sperm/μl) and incubated for 10 min at 21ºC before addition of DMSO or one of the following compounds for 10 min: CK-666 (160 μM), CK-548 (160 μM) KU5593 (100 μM), VE821 (50 μM). Subsequently, the PflMI restriction endonuclease was added to each sample (0.05U/μl) before incubation for an additional 60 min at 21ºC. Extracts were diluted in chromatin isolation buffer (50 mM Hepes-KOH, pH 7.8, 100 mM KCl, 2.5 mM MgCl2) supplemented with 0.125% Triton X-100 and overlaid on top of sucrose cushions (chromatin isolation buffer plus 30% sucrose) in 1.5 ml low-retention tubes (Thermo Fisher Scientific). Samples were spun at 7180 RPM for 30 min at 4ºC in a swing-bucket Sorval rotor (HB-6). Chromatin pellets were resuspended in 10 μl Laemmli buffer, boiled, and fractionated on a 4 – 22% Tris-Glycine gel (Invitrogen) according to standard procedures, followed by transfer of resolved proteins onto PVDF membranes. Following a 1 hr block with 5% milk, membranes were incubated overnight at 4ºC with one of the following primary antibodies against: β-actin (Sigma Aldrich: A5316, 1/1000), Arpc4 (Novus Biologicals: NBP1-69003, 1/500), Capping protein β-2 subunit (Developmental Studies Hybridoma Bank: E00007, 1/10000), Histone H3 (Cell Signaling Technology, 9715, 1/2000) and Xenopus Mre11 34. HRP-conjugated secondary antibodies were used (Anti-Rabbit IgG HRP, Anti-Mouse IgG HRP, Fisher Scientific) and chemiluminescence (Supersignal West Pico Chemiluminescent Substrate, 34077) was used.","divisions":[{"label":"Title","span":{"begin":0,"end":40}}],"tracks":[{"project":"2_test","denotations":[{"id":"29925947-21893598-75110993","span":{"begin":200,"end":202},"obj":"21893598"},{"id":"29925947-21893598-75110993","span":{"begin":200,"end":202},"obj":"21893598"},{"id":"29925947-21893598-75110994","span":{"begin":1606,"end":1608},"obj":"21893598"},{"id":"29925947-21893598-75110994","span":{"begin":1606,"end":1608},"obj":"21893598"}],"attributes":[{"subj":"29925947-21893598-75110993","pred":"source","obj":"2_test"},{"subj":"29925947-21893598-75110993","pred":"source","obj":"2_test"},{"subj":"29925947-21893598-75110994","pred":"source","obj":"2_test"},{"subj":"29925947-21893598-75110994","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#9397ec","default":true}]}]}}