Nuclear and cytoplasmic protein extraction kit

Total protein was extracted using the Lysis Buffer (Thermo Fisher Scientific). The nuclear and cytoplasmic fractions were isolated by the Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN, Nanjing, China) according to the manufacturer's instructions. The supernatant fraction was collected by centrifugation and the protein concentration was determined by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were separated by 10% SDS-PAGE (Epizyme, Shanghai, CHN) at 120 ​V for 80min, and then transferred electrophoretically to a nitrocellulose membrane at 100 ​V for 90min. The transferred membranes were blocked with 5% skim milk for 1 ​h and then incubated with primary antibody including β-catenin (1:2000; Cell Signaling Technology, USA), Lamin B1 (1:2000; Cell Signaling Technology, USA) and GAPDH (1:2000; Cell Signaling Technology, USA) at 4 ​°C for 12 ​h. The membrane was subsequently incubated with second antibody for 1 ​h, and exposed by the FluorChem R system (ProteinSimple, San Jose, CA, USA) for chemiluminescence. LaminB1 and GAPDH were used as the internal control of nucleic protein and total protein, respectively.

Total protein was extracted from RAW264.7 cells using a Whole Cell Lysis assay (Nanjing KeyGen Biotech Co., Ltd.). Total nuclear and cytoplasmic proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction kit (Nanjing KeyGen Biotech Co., Ltd.). Protein concentration was measured using a BCA protein assay kit (Nanjing KeyGen Biotech Co., Ltd.) and 20 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% BSA (Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The membranes were then incubated overnight at 4°C with primary antibodies against NF-κB p65 (1:1,000), p-NF-κB p65 (1:1,000), Nrf2 (1:1,000), HO-1 (1:1,000), NQO1 (1:1,000), NLRC3 (1:1,000), TRAF6 (1:1,000), β-actin (1:2,000) or Histone H3 (1:2,000). Following the primary antibody incubation, the membranes were incubated with an HRP-conjugated anti-rabbit secondary antibody (Abcam; cat. no. ab6721; 1:5,000) at room temperature for 1 h. The membranes were washed multiple times with TBS-Tween-20 buffer and the protein bands were visualized using enhanced chemiluminescence reagent (cat. no. G2020; Wuhan Servicebio Technology Co., Ltd.) and a chemiluminescence imaging system (Bio-Rad Laboratories, Inc.). Densiometric analysis was performed using Image Lab software (version 6.0; Bio-Rad Laboratories, Inc.).