Anti lkb1

The IP assays were performed as described previously.^{39 (link), 49 (link)} Briefly, H9c2 cells, adult cardiomyocytes, or heart tissue were lysed in IP buffer (25 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 1% NP-40; protease and phosphatase inhibitors). Then 300 μg of the lysate was immunoprecipitated overnight at 4 °C with the monoclonal antibodies LKB1 (Sigma-Aldrich) and protein G-agarose (Pierce Biotechnology Ltd., Rockford, IL, USA). The immunocomplexes were probed by western blotting with monoclonal anti-LKB1 (Sigma-Aldrich), SIRT1 (Abcam), SIRT6 (Abcam), or acetyl-lysine antibody (Cell Signaling).

To analyze the protein expression, western blot analysis was performed as previously described.^{47 (link), 49 (link)} The primary antibodies to Nrf2, HDAC1, SIRT1, SIRT6, and AMPK were purchased from Abcam (Cambridge, MA, USA). Anti-LKB1 was obtained from Sigma-Aldrich (Sigma, MO, USA), anti-HO-1, anti-NQO-1, anti-CAT, anti-IKK, anti-IκBα, anti-β-Klotho, anti-CTGF, and anti-TGF-β were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p65, anti-caspase-8, anti-caspase-3, anti-PARP, anti-Bax, anti-Bcl-2, anti-FGFR1, and anti-acetylated-lysine were purchased from Cell Signaling (Danvers, MA, USA). Anti-3-NT was from Millipore (Billerica, CA, USA), and anti-4-HNE was from Alpha Diagnostic International (San Antonio, TX, USA). After three washes with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the membrane was reacted with appropriate secondary antibodies for 1 h at room temperature. Finally, the probed proteins were stained with enhanced chemiluminescence reagent and visualized using the BIO-RAD ChemiDoc Touch Imaging System (BIO-RAD, Hercules, CA, USA).