Acetyl histone h3

ChIP assay was performed with Immunoprecipitation Assay Kits (Millipore) according to the manufacturer's instructions. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at 37°C. The cells were then resuspended in 200 μl of lysis buffer and incubated for 10 min on ice. The lysate was sheared to lengths between 200 and 1000 bp by sonication. The supernatant was pre-cleared with a Salmon Sperm DNA/Protein A Agarose-50% Slurry. The recovered supernatant was incubated with antibodies [Sp1 (1 :10 , polyclonal rabbit IgG, immunogen affinity purified), HDAC1 (1 :10 , monoclonal mouse IgG, protein G purified) and HDAC4 (link) (15 (link) µg, polyclonal rabbit IgG, immunogen affinity purified) (Abcam); acetyl-histone H3 (link) (10 µg, rabbit polyclonal IgG, protein A purified), E2F (link)3 (link) (2 (link) µg, mouse monoclonal IgG, protein A purified) (Millipore)] or an isotype control IgG (15 µg rabbit polyclonal or 5 µg mouse monoclonal IgG, protein A purified) (Millipore) overnight at 4°C with rotation. The antibody/DNA complex was collected using Salmon Sperm DNA/Protein A Agarose Slurry for 1 h at 4°C with rotation, and the complex was eluted by elution buffer. Crosslinks were reversed with 5M NaCl heating at 65°C for 4 h. The DNA sample was then purified and measured by qRT-PCR. The primers are listed in Supplementary Table 4.

Cell lysates were resolved through acrylamide gels using SDS-PAGE and transferred to PVDF-FL membranes (Millipore). Membranes were blocked in Odyssey blocking buffer (Li-Cor) and incubated in specific primary antibodies including MYC (Abcam, 32,072), pAkt (Abcam, 38,449), Acetyl-Histone H3 (Abcam, 4729) and actin (Abcam, 3280). Membranes were incubated with IRDye fluorescent-conjugated secondary antibodies (Li-Cor), and protein expression was detected using Odyssey Infrared imaging system (Li-Cor).

Protein levels from rat kidney cortex homogenates and from cell lysates were quantitated using immunoblotting as described by us previously73 (link)74 (link)75 (link)76 (link)77 (link)78 (link)79 (link)80 (link). The antibodies against HDACs (Cell signalling technology, MA USA), E-cadherin, Fibronectin, Collagen, RAGE (Santa Cruz Biotechnology, USA), TGF-beta, Histone H3, acetyl Histone H3 (Abcam MA USA) and anti-acetyl Histone H4 (Millipore) and, anti-β-actin (Cell signalling technology, MA USA) were used and signals were detected using a chemi-luminiscence-based detection system (Amersham). The levels of protein expression were quatitated using the Quantity one (BioRad) software.