SIRT1 Regulates TSH Release Figure 7. Model for the potential roles of SIRT1 in TSH secretion. Proposed model for regulation of TSH secretion by SIRT1. doi:10.1371/journal.pone.0011755.g007 reaction (PCR)-based protocol [66]. Water and food was provided ad libitum for both groups. Human tissue sections were obtained from the Tokyo Medical and Dental University, and Tokyo Metropolitan Institute of Gerontology. These experiments were approved by the Mitsubishi Kagaku Institute of Life Science Ethics Committee. All experiments were reformed according to protocols approved by the Animal Care and Use Committee of Mitsubishi Kagaku Institute of Life Sciences. Antibodies We used the following antibodies: anti-SIRT1, b-actin, and Flag-M2 (Sigma-Aldrich); PIP5Kc and nucleoporin (BD Trans- duction Laboratories); acetylated lysine (BioVision); GFP and agarose-conjugated GFP (Medical & Biological Laboratories); GAPDH (Millipore, Bedford, MA); ACTH (Nichirei), FSH and GH (DakoCytomation); TOTO-3 (Invitrogen). The anti-TSH, anti-LH, and anti-PRL antibodies were gifted by the Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University. For immunoprecipitation, Kanaho’s lab prepared polyclonal antibodies against mouse PIP5Ka, PIP5Kb, and PIP5Kc [37]. PIP4Kb, prepared in Moses V. Chao’s lab, was detected by using a polyclonal antibody [67]. Secondary antibodies were obtained from Jackson Immunoresearch and Molecular Probes. Protein identification by LC-MS/MS analysis Flag-SIRT1-associated complexes were digested with Achromo- bacter protease I, and the resulting peptides were analyzed using a nanoscale LC-MS/MS system. The criteria for match acceptance have been reported previously [31]. Plasmids and transfection Plasmids encoding Flag-tagged SIRT1 were constructed using the pCMV-Tag2 or pCMV-Tag4 vectors (Stratagene). SIRT1 (H355A) was generated by mutating histidine 355 to alanine [29,30]. Plasmids encoding GFP-PIP5Ks or N-terminally epitope Flag-tagged PIP5Ks were constructed using the pcDNA3-GFP or pcDNA3-Flag (Invitrogen) vectors, as described previously [36]. The QuickChange Site-Directed Mutagenesis (Stratagene) kit was used to generate the PIP5Kc (K265/268R) mutants. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. SIRT1 and PIP5K stealth RNAi were purchased from Invitrogen, and transfections were performed using Lipofectamine 2000 (Invitro- gen) in accordance with the manufacturer’s instructions. Rat TSHb cDNA was generated by PCR. The PCR product was inserted into TagGFP (Evrogen). All constructs were confirmed by sequencing. The expression of TSHb was confirmed by ELISA (Shibayagi), using lysates of GFP-TSHb-overexpressing cells. Immunoprecipitation The transfected HEK293T cells (6-cm dish) were lysed in lysis buffer (25 mM Tris-HCl [pH 8.0], 100 mM NaCl, 2 mM ethylenediamine acetic acid (EDTA) and 0.2% Triton X-100) containing protease inhibitors, 0.5 mM trichostatin A, and 5 mM NAM. After brief sonication, the lysates were cleared by centrifugation. The immunoprecipitates thus obtained were analyzed by immunoblotting. Cytoplasmic proteins obtained from rat pituitary were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents, according to the manufacturer’s instructions (Pierce). Western blot analysis Lysate proteins were separated on an 8% SDS-PAGE gel and then blotted onto polyvinylidene difluoride membranes (Milli- pore). The membranes were blocked using Block Ace (Dainippon Seiyaku) and probed with the appropriate primary antibodies. The bound primary antibodies were detected using the corresponding horseradish peroxidase-conjugated secondary antibodies. The signal was visualized using the ECL kit (GE Healthcare Bio- PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11755