Ab246550

Cardiac fibroblasts (3 × 10⁷) were fixed in 1% formaldehyde and lysed in buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS, protease inhibitor cocktail Set I (539131, Merck Millipore, Billerica, MA, USA)) and sonicated using a epishear multi-sample sonicator, leading to fragments between 300 and 1,000 bp. ChIP was performed using a commercially available ChIP-IT High Sensitivity Kit (53040, Active Motif, Fullerton, CA, USA) according to manufacturer's instructions. DNA-bound protein was immunoprecipitated using an anti-p53 antibody (ab246550, Abcam, Cambridge, UK) and anti-IgG (sc-2025, Santa Cruz, Santa Cruz, CA, USA) as a negative control. The DNA recovered was sequencing and analysed by quantitative polymerase chain reaction (qPCR) to amplified the promoter region of Wnt4: forward 5'-CGCCCGCCTCCGCCGCCAC-3', reverse 5'-CGCAGGGACCGCAGGCACGAA-3', Primers for p53 binding were designed using the SABiosciences'proprietary database (DECipherment of DNA Elements). PCR was performed with equal amounts of specific antibody immunoprecipitated sample, control (IgG) and input. Values were normalized to input measurements and enrichment was calculated using the comparative D-DCt (DDCt) method.

Western blot assays were performed using whole liver lysates and nuclear or microsomal protein lysates from the liver samples. Antibodies against mouse (# NB100-2522) or human (# D8012) SIRT6 were purchased from Novus Biologicals (Centennial, CO, USA) and Cell Signaling (Danvers, MA, USA), respectively. Antibodies against mouse P53 (# ab26 and # ab131442), human P53 (# ab246550), and Tubulin (# ab4074) were purchased from Abcam (Cambridge, Cambridgeshire, UK). Antibodies against human P53 (# SC-126) or Histone (# SC-517576) were purchased from Santa Cruz (Dallas, TX, USA). Antibodies against CYP7A1 (# TA351400) or CYP8B1 (# TA313734) were purchased from Origene (Rockville, MD, USA). The antibody against calnexin (#NB100-1965) was purchased from Novus (Centennial, CO, USA). Peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Primary antibodies were diluted at 1:1000 except for the Tubulin antibody, which was diluted at 1:5000.

The total proteins from N2A cells were lysing with 1% SDS. Subsequently, the extracts were separated using SDS-PAGE on 10% gel and electro-transferred onto PVDF membranes (Bio-Rad, USA), then blocked with 5% nonfat milk at room temperature. The blots were incubated with the primary antibodies P53 (1:1000, abcam, ab246550) and Bax (1:1000, abcam, ab216494) overnight at 4 ℃. On the second day, the blots were incubated with secondary antibodies (4050–05 or 1031–05, Southern Biotech) for 1 h at room temperature, and visualized on amersham Imager 600. Data was normalized to β-actin and quantified using Image J software (NIH, Bethesda).