Anti sirt1 h 300

Antibodies for P-AMPK (Thr¹⁷²/Ser^485/491), P-Akt (Ser⁴⁷³⁾, P-GSK3β (Ser⁹), total AMPK, ACC and CAMKKβ were obtained from Cell Signaling (Danvers, MA) and P–ACC (Ser⁷⁹) from EMD Millipore (Billerica, MA). Rabbit polyclonal anti-SIRT1 (H-300) was from Santa Cruz Biotechnology (Santa Cruz, CA). “SAMS” peptide and the polyclonal antibody used for immunoprecipitation of AMPK’s α2 catalytic subunit were obtained from QCB biotechnology (Hopkinton, MA). [γ-³²P] ATP was obtained from Perkin-Elmer (Boston, MA) and Protein A/G plus conjugate from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were purchased from either Sigma-Aldrich or Fisher Scientific.

HEK293T or HEK293 cells were transfected with an expression vector as indicated in the figure. 24 h after transfection, the cells were harvested in cell lysis buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1% TritonX-100) supplemented with protease inhibitor mixture (Roche Diagnostics). Whole cell lysates (500 μg) were immunoprecipitated with 10μl of M2 beads (A2220; Sigma) or 2 μg of antibodies as indicated. Immunoprecipitates were washed 5 times with cell lysis buffer and resolved by SDS-PAGE gel followed by Western blot with the indicated antibodies. Antibodies used were: anti- FLAG^® M2 Affinity Gel (M2; Sigma), anti- PPARγ (H100 and E8), anti-SIRT1 (H-300), anti-cyclin E (M-20), anti-aP2 (C-15), anti-β-actin (C4) (Santa Cruz), and anti-cyclin D1 (DCS-6; Santa Cruz or Ab3; NeoMarker). The abundance of immunoreactive protein was quantified using a densitometer (Image Quant version 1.11, Molecular Dynamics Computing Densitometer, Sunnyvale, CA).