Protease and phosphatase inhibitors

293 T WT or ARH3^−/− cells were plated with 0.5 μM Olaparib or 5 μM Veliparib, cultured for three days and transfected in the presence of the drug using Polyfect (Qiagen) with a plasmid expressing H3-GFP for 24 h. The cells were washed with PBS and lysed with Triton X-100 lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1% Triton X-100) supplemented with 5 mM MgCl₂, protease and phosphatase inhibitors (Roche), 1 μM PARG inhibitor and 1 μM Olaparib. The lysates were incubated with Benzonase (Sigma) for 20 min at 4 °C, centrifuged at 20,000 × g for 15 min, and the supernatants were collected. Protein concentrations were normalized, then the cell lysates were incubated with GFP-Trap MA magnetic agarose beads (ChromoTek) for 2 h while rotating at 4 °C. The beads were washed several times with Triton X-100 lysis buffer and eluted with 2x NuPAGE LDS sample buffer (Invitrogen) with TCEP (Sigma). The samples were then analysed by Western Blotting.

The impact of CLP treatment on the expression and phosphorylation of relevant proteins in liver tissues was examined by Western blotting. In briefly, each liver tissue sample was homogenized in RIPA buffer reagent containing protease and phosphatase inhibitors (Qiagen). The tissue lysates (30 µg/lane) were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis on 10% gels and transferred onto nitrocellulose membranes (Bio-Rad, USA). After being blocked with 5% skimmed dry milk in TBST buffer, the membranes were reacted with anti-PPAR-α (D161086, Sangon, China), anti-MCAD (GB112107, Servicebio, China), anti-CPT-1 (sc-393070), and GAPDH (sc-47724, Santa Cruz Biotech, USA) overnight at 4 oC. After being washed, the bound antibodies were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) and observed with enhanced chemiluminescent plus reagents (Amersham Biosciences, USA). The data were quantitatively analyzed using the Image-Pro Plus software.