Anti iκbα

CRC cells or liver metastatic tissues were lysed with RIPA Lysis Buffer (Beyotime, China) containing 1% PMSF (Beyotime). Besides, the nuclear protein was isolated by the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein from different conditions were separated by 6–14% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk or 1% BSA, the membranes were incubated with following primary antibodies at 4 °C overnight: anti-E-cadherin (1:1000, 60,335–1-Ig, Proteintech, China), anti-N-cadherin (1:1000, 66,219–1-Ig, Proteintech, China), anti-fibronectin (1:500, 15,613–1-AP, Proteintech, China), anti-p-IκBα (1:500, bs-2513R, Bioss, China), anti-IκBα (1:500, bs-1287R, Bioss, China), anti-p65 (1:500, 10,745–1-AP, Proteintech, China), anti-β-actin (1:500, KGAA001, KeyGen, China) and Histone H3 (1:2000, AM8433, ABGENT, USA). After rinsed with TSBT, the membranes were incubated with their corresponding secondary antibodies at 37 °C for 45 min. The protein bands were visualized using the ECL reagent (Beyotime).

GA was purchased from Meilun Biological Co., Ltd. (Dalian, China), and dissolved in 10% DMSO at a concentration of 5 mg/mL. The chemical structure of GA is shown in Figure 1. The antibodies used for western blotting and immunohistochemistry were as follows: anti-Bcl-2 (Boster, China), anti-Bax (Boster, China), anti-IκBα (Bioss, China), anti-cleaved caspase-3 (Abcam, USA), anti-NF-κB (Boster, China), anti-p-NF-κB (Bioss, China), anti-Cox-2 (Boster, China), anti-iNOS (Bioss, China), anti-β-actin (Boster, China), and anti-Histone H3 (Bioss, China).