, 2008). Two days later, cells were stimulated with indicated agents. Neurons were fixed in 4% paraformaldehyde/2% sucrose in 1X PBS for 20 min at room temperature, permeabilized, and stained with indicated primary and secondary antibodies (see Supplemental Experimental Procedures). The localization of HDAC5 was categorized as cytoplasmic, nuclear, or both (evenly distributed across nucleus and cytoplasm) for each neuron under experimenter-blind conditions. C57BL/6 mice (Charles River) were injected once per day (intraperitoneally [i.p.]) with saline or cocaine (5 or 20 mg/kg) before rapid isolation of brain tissues at indicated times
Fasudil molecular weight after injection. HDAC5 was immunoprecipitated from diluted total striatal lysates and analyzed by standard western blot analysis with indicated antibodies (see Supplemental Experimental Procedures for dilutions and sources). Cytosolic and nuclear extracts were prepared with NE-PER nuclear and cytoplasmic extraction kit (Pierce Biotechnology) according to the manufacturer’s
instructions. HEK293T cells were cultured in Dulbecco’s modified MG-132 purchase Eagle’s medium containing 10% (v/v) FBS, penicillin-streptomycin (1X; Sigma-Aldrich), and L-glutamine (4 mM; Sigma-Aldrich). HEK293T cells were transfected with HSV-flag-hHDAC5 using calcium phosphate and harvested 2 days after transfection. Flag-HDAC5 was prepared from HEK293T cell extracts in RIPA buffer (50 mM Tris [pH 7.4], 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholates, 10 mM NaF, 10 nM
okadaic acid, and complete protease inhibitor cocktail tablet [1X; Roche]) by IP with anti-flag antibody (M2)-conjugated beads. The protein was separated by SDS-PAGE and stained with Coomassie brilliant blue. The HDAC5 band was excised from the gel, washed, and then digested with trypsin. The tryptic digests were analyzed with an EC-MS/MS system. Flag-HDAC5 was prepared from transfected HEK293T cell extracts by IP with anti-flag antibody (M2)-conjugated beads in RIPA buffer. For the PKA phosphorylation, immunoprecipitated beads were washed and suspended also in PKA phosphorylation buffer (50 mM PIPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, and protease inhibitor) and incubated with or without recombinant PKA catalytic subunit (Sigma-Aldrich) or alkaline phosphatase (Roche) in the presence of 1 mM ATP at 30°C. For the Cdk5 phosphorylation assay, the immunoprecipitated flag-HDAC5 on the beads was washed and resuspended in alkaline phosphatase buffer (Roche) and incubated with alkaline phosphatase at 37°C for 2 hr. Dephosphorylated beads were washed with RIPA buffer three times and Cdk5 kinase assay buffer (10 mM MOPS [pH 7.2], 10 mM MgCl2, 1 mM EDTA) three times, and the immunoprecipitated HDAC5 was incubated with or without Cdk5-p25 (Sigma-Aldrich) in the presence of 1 mM ATP at 30°C.