Anti histone h3 monoclonal antibody

T98G cells were treated with FAEW and fluoxetine for 24, 48, and 72 h, MG-132 for 1 h, followed by TNF-α induction for 3 h. MG-132 was used as positive control drug. Nuclear protein extracts were prepared according to the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific, USA) supplemented with EDTA-free Halt Protease Inhibitor Cocktail (Thermo Scientific). Protein concentrations were measured with Nano drop 1000 spectrophotometry (Thermo Scientific). The densities of the protein bands were quantified by FluorChemQ software (Biozym Scientific Company, Oldendorf, Germany). Nuclear p65 levels were determined with an anti-NF-κB monoclonal antibody (1:3000, Cell signaling). Histone H3 protein levels served as the internal control, using the anti-Histone H3 monoclonal antibody (1:3000, Cell Signaling). The inhibition effects of FAEW and fluoxetine toward p65 protein expression were calculated by comparison with untreated TNF-α induction group after using the control group without TNF-α for the quantification of western blots.