Nuclear and cytoplasmic extraction reagent kit

Nuclear and cytoplasmic proteins were extracted from HBZY-1 cells using the Nuclear and Cytoplasmic Extraction Reagent Kit (Beyotime Biotechnology, Shanghai, China). The concentration of each protein sample was determined by BCA reagent (Beyotime Biotechnology, China). The total protein was separated by 12% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA (w/v) for 1 h at room temperature, the membranes were incubated with primary antibodies for COX-2, iNOS, nuclear factor kappa B (NF-κB), inhibitor alpha (IκBα), phosphorylated IκBα (p-IκBα), IκB kinase beta (IKKβ), phosphorylated IKKβ (p-IKKβ) and p65, respectively, overnight at 4 °C and then incubated with secondary antibodies at room temperature for 1 h. Immunoreactive bands were visualized by ECL kit (Millipore), and the densitometry was analyzed by ImageJ software (NIH, Bethesda, MD, USA).

Total cell lysates were prepared using RIPA lysis buffer added PMSF (Solarbio, China). Cytoplasmic and nuclear TAZ protein were extracted separately using nuclear and cytoplasmic extraction reagent kit (Beyotime Bio Corp, China). Protein concentration was determined using BCA protein assay kit (Beyotime Bio Corp, China). Proteins (20 μg) were resolved by SDS-PAGE and electrotransfered to PVDF membranes (Millipore, USA). Appropriate primary antibodies (Santa Cruz, CA, USA) were used for WB analysis.

Western blotting analysis was performed as reported previously (Hong-Tao Wang, Neurotox Res 2017, 31:505–520). The hippocampi were removed and snap-frozen in liquid nitrogen. Nuclear extracts were collected using Nuclear and Cytoplasmic Extraction Reagent Kit (Beyotime Biotechnology, China) according to the manufacturer’s protocol. Thirty micrograms of protein from each sample was loaded onto and separated by a 15% Bis-Tris Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis gel. The blotted proteins were transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight with the following antibodies: rabbit polyclonal anti-histone H3 antibody (Servicebio; dilution, 1:30000) and rabbit polyclonal antibody H3K9/14ac (Origene; dilution, 1:1000). Then, the membranes were washed and incubated with horse-raddish peroxidase conjugated secondary antibodies (CWBIO; dilution, 1:1000) for 2 h at room temperature. After three rinses (10 min in each) in tris-buffered saline with 0.1% Tween-20, immunoreactivity was detected with an ECL Western Blotting Detection Kit (CWBIO, China). Results were standardized to histone H3 control protein.