Cell lysates were analyzed by the dual luciferase assay (Promega) on a luminometer. To assess the activity of IKK, IKK was immunoprecipitated by IKKα antibody and protein G-Sepharose, and the assay was performed at 30°C for 1 hour in buffer
containing 20 mM Tris HCl, pH 7.5, 20 mM MgCl2, 2 mM dithiothreitol, 20 μM ATP, 2 μg GST-IκBα, and [γ-32P]ATP. The reaction was stopped by addition of Laemmli buffer and was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by a transfer onto a membrane for imaging. Whole cell extracts were prepared as described.8 Equal amounts of Galunisertib molecular weight the extract (20 μg) were separated by 8%-15% SDS-PAGE and the proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). MeCP2, type I collagen, and β-actin were detected by incubating with rabbit polyclonal anti-MeCP2 (1:1,000) (Abcam), anti-type I collagen (1:4,000), and anti-β-actin (1:5,000) primary antibodies (Santa Cruz Biotechnology) in TBS (100 mM Tris-HCl,
1.5 M NaCl, pH 7.4) with 5% nonfat milk overnight at 4°C followed by incubation with horseradish peroxidase-conjugated goat antirabbit secondary antibodies (1:4,000) (Sigma) at room temperature for 2 hours. The antigen-antibody complexes’ chemiluminescence was detected using the ECL detection kit (Pierce). Talazoparib cell line For assessing Pparγ epigenetic regulation, carrier ChIP was performed using Raji cells as the source of carrier chromatin. For Galeterone native ChIP, 20 μg of HSC chromatin was mixed with 80 μg of Raji cell chromatin. For crosslink ChIP, Raji cells (1.4 × 107 cells) were mixed with HSCs (0.2 × 106 cells) and fixed with 1% formaldehyde on the rotating platform for 5-10 minutes at room temperature followed by addition of glycine to a final concentration of 0.125 M. After lysis of the cells with SDS buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) with protease inhibitors, the
lysates were sonicated and snap-frozen in aliquots. For chromatin IP, diluted samples were first precleared using protein G-agarose beads and then incubated with antibody against Ser2P RNApolyII, MeCP2, H3K27me2, H3K4me2, and H3Kacetylated (Abcam) at 1 μg/μL at 4°C overnight followed by precipitation with protein G-agarose beads. After elution of immunoprecipitated complex, crosslinking was reversed with 5 N NaCl and proteins digested with protease K. Extracted chromatin was subjected to real-time PCR using the primers flanking a segment within Pparγ promoter or exon as described.17 Ct values of the samples with nonimmune IgG were subtracted and compared to their respective input Ct values. The aqueous YGW extract (350 mg/mL in PBS) was applied to size exclusion chromatography using Super Prep Grade gel in XK 16/70 column (Amersham Pharmacia Biotech, Piscataway, NJ) and PBS as a mobile phase solvent.
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