Nucleoprotein and cytoplasmic protein extraction kit

Frozen tissues were homogenized and proteins were extracted using a nucleoprotein and cytoplasmic protein extraction kit (Keygen Biotech, China); 30 μg of protein was mixed with SDS sample buffer. Proteins were separated on standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8–10% gels) then transferred onto 0.45-μm polyvinylidene fluoride membranes (Millipore, United States). Membranes were blocked in 5% milk for 1 h and incubated overnight at 4°C with the following primary antibodies: mouse anti-HDAC2 (1:600, Abcam, United States), rabbit anti-GAPDH (1:500, Goodhere, China), and mouse anti-β-tubulin (1:1000, Beyotime, China). Membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, Jackson, United States) for 1.5 h at a room temperature about 25°C. Proteins were detected using a ChemiDoc luminescence system (Bio-Rad).

The cytoplasmic and nuclear proteins were extracted using the nucleoprotein and cytoplasmic protein extraction kit (KeyGEN Biotech, Jiangsu, China). The nuclear protein was used to detect the expression of p65, p53, Bax, p-JNK and JNK, and the total protein was extracted using highly efficient RIPA tissue/cell rapid lysate (Solaibio, Beijing, China) to test the expression of p65, p53, Bax, p-JNK and JNK and actin. Protein samples (30 µg) were loaded on a 12% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred to a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked with blocking buffer for 1 h, then incubated with the following primary antibodies: p65 (CST, Boston, USA), p53 (CST, Boston, USA), Bax (CST, Boston, USA), p-JNK (CST, Boston, USA), JNK (CST, Boston, USA), actin (Santa Cruz, Dallas, USA), and proliferating cell nuclear antigen (Affinity Biosciences, Ohio. USA) at 4°C overnight. It was then incubated with secondary antibodies for 1 h.

Western blotting was performed according to our previous protocols (Liu et al., 2015 (link), 2016 (link); Qiu et al., 2016 (link)). Bilateral hippocampus (n = 3 in each group) were dissected and sonicated in ice cold lysis buffer (P10013B, Beyotime Institute of Biotechnology, China). The lysate was centrifuged at 12,000× g for 10 min at 4°C. Then the supernatant fraction was collected for Western blotting analysis. Nucleoprotein and cytoplasmic protein were extracted with Nucleoprotein and Cytoplasmic Protein Extraction Kit (KeyGEN BioTECH, China). BCA Protein Assay Kit (T9300A, Takara, Shiga, Japan) was used to detect the protein concentration. The primary antibodies used in Western blotting analysis were as follows: Rabbit Anti-Nuclear factor-κB (NFκB; P105/P50; 13586S, 1:1000; Cell Signaling, Chicago, IL, USA), Mouse Anti-NFκB (P65; 6956S, 1:1000; Cell Signaling, Chicago, IL, USA), Rabbit Anti-inhibitor of NF-κB α (IκBα; 4814S, 1:1000; Cell Signaling, Chicago, IL, USA), Rabbit Anti-P-IκBα (9246S, 1:1000; Cell Signaling, Chicago, IL, USA), Rabbit Anti-GAPDH (14C10; 2118S, 1:1000; Cell Signaling, Chicago, IL, USA). The secondary antibodies were Anti-Rabbit/Mouse IgG, HRP-linked antibody (7076/7074, 1:2000, Cell Signaling, Chicago, IL, USA). The target protein bands were quantified by using FluorChem Q system (ProteinSimple, San Jose, CA, USA).