Phosphatase inhibitor

Whole cell lysate protein was prepared from the above treated cells by using ice-cold radio immunoprecipitation assay buffer (Active Motif, Carlsbad, CA) containing a complete protease inhibitor cocktail (Roche, Basel, Schweiz) and phosphatase inhibitor (Active Motif). A standard procedure of western blotting was performed as described previously^{27 (link)}. Briefly, after determination of protein concentration with Bradford assay, 20 µg protein of each sample was electrophoresed in SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Merck Millipore). After blocking and incubation with primary antibodies (Table 1), the membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (1:5000; Proteintech). The peroxidase activities of target protein bands were detected using an enhanced chemiluminescent detection system (Merck Millipore) and visualized by using a G-Box capture system (Syngene, Cambridge, UK). The abundance of phosphorylated PDHE1α was expressed as the ratio over total PDHE1α. The abundance of PDK4 and 11β-HSD2 was expressed as the ratio over the housekeeping gene α-Tubulin.

The presence of physical interaction between HDAC1 and DAXX was examined in non-transfected HiB5 and HiB5 cells transduced with LVVs carrying non-target RNA or HDAC1 shRNAi. Cell culture dishes were placed on ice, washed three times with cold PBS, scraped out in 1 ml of ice-cold PBS containing phosphatase inhibitors (Active Motif, Carlsbad, CA, USA), transferred to a 1.5 microcentrifuge tube and centrifuged for 3 min at 4 °C at 500 g. Nuclear protein extraction and co-immunoprecipitation procedures were performed using the Nuclear Complex Co-immunoprecipitation kit (Active Motif) according to the manufacturer's protocol. Nuclear proteins (200 μg) were immunoprecipitated by overnight incubation with a chromatin immunoprecipitation-validated rabbit anti-HDAC1 antibody (2ug; 40967 Active Motif), or rabbit IgG (Jackson Labs, West Grove, PA, USA) used as negative control at 4 °C. Protein–antibody immune complexes were separated using protein G magnetic beads (Active Motif) for 1 h at 4 °C, washed (four times) and re-suspended in 20 μl of 2x Loading Buffer containing DTT (Invitrogen) for western blot analysis. Co-precipitated DAXX was detected using a rabbit anti-DAXX antibody at 1:500 (Santa Cruz).

Whole cell extracts were prepared using RIPA Buffer (Cell Signaling, Leiden, Netherlands) including Protease Inhibitor Cocktail (Sigma-Aldrich, Munich, Germany). Prior to short-term phosphorylation studies, cells were rested in basal media without supplements for 60 min. Before lysis, cells were washed twice with ice-cold PBS containing phosphatase inhibitors (Active Motif, Carlsbad, CA, USA). Protein concentration was determined by BCA Protein Assay Reagent (Perbio Science, Bonn, Germany/Thermo Fisher Scientific, Waltham, MA, USA) to ensure equal amounts of protein used for subsequent analysis. Proteins were denaturated for 10 min at 95 °C, separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with the following antibodies: HMOX1 and eNOS (BD Transduction Laboratories, Heidelberg, Germany); NRF2 (Abcam, Cambridge, UK); NQO1, ACTB as a loading control, P-eNOS (Ser1177), AKT and P-AKT (Ser473) (Cell Signaling, Leiden, The Netherlands) for 1 h. Afterwards, membranes were incubated for 1 h with anti-rabbit (Acris, Herford, Germany) or anti-mouse (Life Technologies, Darmstadt, Germany) IgG HRP-conjugated secondary antibodies. Protein expression was detected with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Rodgau, Germany) and quantified using AIDA Image Analyzer software (Raytest, Berlin, Germany).