Identification of CBP-interacting proteins by LC-MS/MS

Published: 26-10-2019| Version 2 | DOI: 10.17632/gh228m9gms.2
Contributor:
Yasuhiro Funahashi

Description

To concentrate and identify transcriptional factors that are associated with reward-related learning and memory, we injected cocaine into mice and placed them into the conditioned place preference (CPP) test chamber for 30 min. Brains from 10 mice were rapidly removed and coronal slices (1 mm) were produced using a mouse brain slicer matrix (Brain Science Idea). The striatum and NAc (approximately +1.54 mm from Bregma) were collected using 3 mm diameter biopsy punch (Miltex). Collected tissues were homogenized with Hypotonic buffer (Nuclear Extract Kit, Active Motif) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (PhosStop, Roche) using potter homogenizer and incubated on ice for 15 min. The lysate was centrifuged at 800×g for 10 min at 4°C. The pellet was resuspended with Hypotonic buffer and incubated on ice for 15 min. After addition of a detergent and vortexing for 10 sec, the lysate was centrifuged at 16,000×g for 2 min. The nuclear pellet was extracted in lysis buffer [20 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail and PhosStop, pH 7.5], and then sonicated 3 times for 10 sec. The lysate was centrifuged at 16,000×g for 10 min at 4°C. The supernatant was used as the nuclear extracts. A total of 250 pmol of GST or GST-CREB binding protein (CBP)-N-terminal transactivation domain (N-TAD), immobilized on glutathione Sepharose 4B beads, was incubated with the nuclear extracts for 1 hr at 4°C with rotation. The beads were then washed three times with lysis buffer and an additional three times with wash buffer (20 mM Tris/HCl, 1 mM EDTA, and 150 mM NaCl, pH 7.5) to remove the detergent from the beads. The bound proteins were extracted from the beads using urea solution, reduced via incubation in 5 mM dithiothreitol for 30 min, and alkylated using 10 mM iodoacetamide for 1 hr in the dark. The proteins were digested with Trypsin/Lys-C (Promega). Demineralization was performed using SPE c-tips (Nikkyo Technos) according to the manufacturer’s instructions. The peptides were analyzed by LC−MS using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc). To select proteins that specifically interact with CBP, the proteins from the control GST column were removed from the data. High-abundance proteins (such as keratin and ribosomal protein) were also excluded. We identified more than 400 proteins that specifically interact with GST-CBP-N-TAD but not with GST.

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