Tata box binding protein tbp

Protein concentration was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal concentrations of proteins were mixed with SDS sample buffer and denatured at 95 °C for 5 min. The samples were resolved with 8% SDS–page gels which were then transferred onto nitrocellulose membranes. The membranes were blocked with 5% fat-free milk in 0.1% Tris-buffered saline with Tween (TTBS) for 2 hours and then probed with primary antibodies: anti-Nrf2 antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), anti-NLRP3 antibody (Cell Signaling Technology, Beverley, CA) at 4 °C overnight. After being washed for three times with TTBS, the membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) in TTBS at room temperature for 2 hours. Membranes were washed again with TTBS three times and then visualized on X-ray films using a chemo-luminescence detection system (ECL, GE Healthcare). β-actin (Santa Cruz Biotechnology) and TATA box binding protein (TBP) (Abcam, Cambridge, UK) were used as protein loading control for cytoplasmic and nuclear proteins, respectively. The relative band intensities were measured by image analysis software Gel-Pro Analyser.

Luteolin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, 2′,7′-dichlorofluorescein diacetate (DCF-DA), a mouse/rabbit red starter duolink kit, and propidium iodide (PI) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Luteolin was prepared in 100% dimethyl sulfoxide to obtain a 100 mg/ml stock solution, which was aliquoted and stored at −20 °C for 3–6 months for use in the various experiments. Primary antibodies against p53, p21, Bcl-2, Bax, caspase-9, glutamate cysteine ligase catalytic (GCLc), glutathione synthetase (GSS), catalase, heme oxygenase-1 (HO-1), Nrf2, TET1, TET2, TET3, DNMT3B, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DNMT1, phospho-Nrf2, and TATA box-binding protein (TBP) were purchased from Abcam (Cambridge, MA, USA), and caspase-3 and DNMT3A were purchased from Cell Signaling Technology (Beverly, MA, USA).