MassIVE MSV000089825

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Calcineurin signaling regulates the formation of activity-induced DNA breaks by dephosphorylating Topoisomerase IIB at the nuclear periphery

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Description

994820 Fear conditioning: Top2B immunoprecipitated from mouse hippocampus dissected 30 minutes after Fear Conditioning 994821 Control: Top2B immunoprecipitated from hippocampus dissected from mouse that were in home cages. Complete description: Hippocampus brain tissue (4 mouse per sample) from were lysed in in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% N-Lauroylsarcosine, phosphatase and protease inhibitors cocktail) by passing through a 25-gauge needle and incubated with Benzonase Nuclease (>=250 units/microliters) 2microliters for 0.3ml of sample for 3hrs followed by sonication. Samples were centrifuged at 20000 x g for 5 min. The supernatants were collected, and protein concentrations determined using the BCA assay. Equal amounts of protein and 6 microliters of Top2B antibody (Rabbit, Abcam, ab125297) per sample were incubated overnight. Samples were incubated with 25 microliters of protein A/G-magnetic beads for 3 h and the beads were washed 4 times with high salt buffer (500 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS) and one more time with TBS with 0.1% Triton X-100. All procedures were performed at 4C. Beads were re-suspended in Laemmli buffer to separate proteins by electrophoresis.The gel was stained with Coomassie blue and the Topoisomerase 2 band was cut. [doi:10.25345/C5639K90W] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Topoisomerase 2B ; Calcineurin ; NMDA ; KCl ; phosphorylation

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Principal Investigators:
(in alphabetical order) 		Ram Madabhushi, UT southwestern medical center, United States
Submitting User: jjotto
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