Sirt1 sc 15404

Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin. Protein concentration was measured by BCA Protein Assay (Pierce). Total protein (10 μg) was resolved on 10% SDS-PAGE, transferred to PVDF membranes (Immobilon-P, Millipore), and probed with primary antibodies against MTA2 (sc-55566), HDAC1 (sc-7872), SIRT1 (sc-15404) and p53 (sc-126) (Santa Cruz); YY1 (#2185), p21 (#2946), PUMA (#4976), CDK4 (#2906), CDK6 (#3136) and FAS (#8023) (Cell Signaling); MDM4 (mAb 8C6) and MDM2 (mAb 2A10) (EMD Bioscience); or anti-HA (clone 3F10, Roche), followed by incubation with horseradish peroxidase-(HRP) conjugated sheep anti-mouse or anti-rabbit Ig (Amersham). Signals were developed using SuperSignal West Femto (Pierce). Membranes were stripped and reprobed using α-tubulin mouse mAb (B-5-1-2, Sigma) or β-actin (JLA20, Millipore). Immunoblots were quantified by densitometry using VersaDoc MP 4000 (Bio-Rad).

Following treatment with SPD, H9C2 cells were seeded onto glass cover slips in 24-well plates. For detection of PGC-1α and SIRT1, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with PBS/0.1% Triton X-100, blocked with 3% BSA in PBS, and incubated with PGC-1α (sc-518038) and SIRT1 (sc-15404) antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. Fluorescent secondary antibodies (ab150113 and ab150075; Abcam, Cambridge, MA, USA) were used for detection according to the manufacturer’s instructions. Immunofluorescence was assessed with a confocal microscope (Carl Zeiss LSM510, Tokyo, Japan). Confocal laser scanning microscopy analysis of PGC-1α and SIRT1 colocalization was performed for H9C2 cells.