Nuclear and cytoplasm extraction kit

Using a commercial available Nuclear and Cytoplasm Extraction Kit (Active Motif), nuclear proteins were isolated from left ventricles of rats or primary neonatal rat ventricular cardiomyocytes. The homogenate proteins were separated on SDS-PAGE and then transferred to PVDF membrane (Millipore). The membranes were incubated with primary polyclonal antibodies against p65 (Cell Signal Technology), IκB-α (Cell Signal Technology), Histone-H3 (Sigma), p-mTOR (Cell Signal Technology), mTOR (Cell Signal Technology), PPARα (Sigma), PGC-1α (Calbiochem), GAPDH (Sigma), and α-tubulin (Sigma) overnight at 4°C. After washing in TBS-T buffer, the second antibodies (Promega) were conjugated at room temperature, and then protein bands were visualized using enhanced Super-Signal West Pico substrates (Pierce).

For immunoprecipitation and Western blotting [50 (link)], mouse anti-PARP-1 polyclonal antibody, mouse pan-Acetylation monoclonal antibody and mouse β-Actin monoclonal antibody was purchased from Proteintech (Proteintech Group, Chi cago, IL, USA). Mouse anti-PAR-monoclonal antibody was purchased from Trevigen (Trevigen Inc., Gaithersburg, Maryland, USA). Rabbit anti-sirtuin 3 (SIRT3) polyclonal antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and Proteintech (Proteintech Group, Chi cago, IL, USA). Nuclear proteins were extracted with a commercially available Nuclear and Cytoplasm Extraction kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s recommendations. Western blot analyses were performed as previously described [19 (link), 51 (link)] and β-Actin was used as a loading control. For co-IP, total proteins (400μg) incubated with 1μg anti-PARP-1 antibody for overnight (mouse normal IgG was used as a control), or nuclear proteins (200-300μg) incubated with 1 μg anti-SIRT3 antibody for overnight (rabbit normal IgG was used as a control), followed by 4h incubation with protein A/G PLUS-Agarose (Santa Cruz, CA, USA) at 4°C. The co-IP proteins were detected by Western blot.

Using a commercial available Nuclear and Cytoplasm Extraction Kit (Active Motif), nuclear proteins were isolated from left ventricles of rats or NRVMs. The proteins (20 μg) were separated by SDS-PAGE on 10% separation gels, transferred to Hydrophobic Polyvinylidene (PVDF) membranes, and blocked with TBST (Tris-buffered saline with Tween-20) containing 5% non-fat dry milk. Primary antibodies of p65 (1:500 dilution), PGC-1α (1:1000 dilution), ATP5D (1:1000 dilution), β-actin (1:500 dilution) and lamin B (1:1000 dilution) were incubated at 4°C overnight. Then membranes were incubated with horseradish peroxidase-linked secondary antibodies (anti-rabbit, anti-mouse IgG). Detection was performed with chemiluminescence reagents (Amersham Biosciences). The results were quantified by the Quantity One software (Bio-Rad Laboratories, Hercules).