, Wood Dale, IL, USA, cat# 51386). The

brain was sliced using standard razor blades (GEM, Personna American Safety Razor Co., Cedar Knolls, NJ, USA; cat# 62–0165) into 2 mm slices. PVN were either quickly frozen for quantitative PCR or immediately fixed in 2.2% formaldehyde for the ChIP analyses. The PVN was punched using a microdissecting needle of an appropriate size. In situ hybridization and immunostaining were performed as we previously described (Blechman et al., 2007). Total RNA was prepared from fish larvae and mouse PVN tissues using the TRI-REAGENT (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. RNA preparations were treated with DNase recombinant I, RNase free (Roche, Basel, Switzerland) to avoid contamination of genomic DNA. First-strand cDNA was synthesized from 0.5–1 μg of total RNA PI3K inhibitor using an oligo(dT)15 primer and SuperScript II reverse transcriptase (RT) (Invitrogen, Blackrock, Co. Dublin, Ireland). The PCR was performed

in 96 plates using a 7300 real time PCR system (Applied Biosystem, Foster City, CA, USA). The total cDNAs were amplified using a SYBR Green qPCR kit Abiraterone clinical trial (Finnzymes, Espoo, Finland) according to the manufacturer’s instructions. The relative quantity (RQ) was calculated using the Relative Quantification program (Sequence Detection System software version 1.2.2) according to the comparative method. Samples were normalized according an endogenous gene (β-actin for zebrafish very fish or hprt for mouse) and to a control time zero sample, which served as a calibrator. ChIP analysis was performed on 6-day-old larvae (pools of 50 larvae per treatment) or adult mouse PVNs (pools of five punches per treatment) as described in Blechman et al. (2011). For IP, we used 4 μl of affinity-purified anti-Otp, 1 μl of anti-Pol II, or control Rabbit IgG. Sequential ChIP-reChIP analysis was performed using a previously described

protocol (Furlan-Magaril et al., 2009). Proteins for IP were extracted from a pool of 100 fish larvae per treatment as we previously described (Blechman et al., 2007). IP was performed using affinity-purified anti-Otp antibody, which was covalently bound to an Affi-Gel 10 beads (Bio-Rad, Hercules, CA, USA) and incubated with the protein extracts for 3 hr at 4°C. Thereafter, beads were extensively washed and protein complexes were eluted (200 mM Glycine-HCl pH 2.7, 150 mM NaCl) for 15 min at room temperature. The resulting protein complexes were subjected to 12% SDS-PAGE under nonreducing conditions, followed by immunoblotting with an antibody directed to phospho-CREB(Ser133). Nitrocellulose membranes were stripped and reblotted with an anti-Otp antibody. Pools of 6-day-old zebrafish (ten larvae) were used for cortisol measurements.

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