Horseradish peroxidase conjugated goat anti rabbit igg h l secondary antibody

Total protein was extracted from the tissues using RIPA protein lysis buffer (Beyotime Institute of Biotechnology). Nuclear and cytoplasmic proteins were separated using the Nuclear and Cytoplasmic Protein Extraction Kit. The protein concentration was determined using a BCA protein concentration kit (Beyotime Institute of Biotechnology) Total protein (50 µg/lane) was separated by SDS-PAGE on 10% gels and then transferred onto PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: NF-κB p65 (1:1,000; cat. no. ab16502; Abcam), NF-κB inhibitor α (IκBα; 1:1,000; cat. no. ab32518; Abcam), phosphorylated (p)-IκBα (1:1,000; cat. no. ab133462; Abcam) and β-actin (1:5,000; cat. no. ab8226; Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:5,000; cat. no. AS014; ABclonal Biotech Co., Ltd.) at room temperature for 1 h. Lamin B1 antibody (cat. no. ab16048; Abcam) was used to detect lamin B1 as a housekeeping protein in the nuclear fraction. Protein bands were visualized using an enhanced chemiluminescence detection kit (ECL Plus; EMD Millipore) and measured using Image J 1.47 software (National Institutes of Health).

The total protein was extracted from C-33A and CaSki cells by RIPA buffer (Beyotime Institute of Biotechnology). The concentration of protein was detected by BCA kit (Beyotime Institute of Biotechnology). Subsequently, protein samples (20 µg/per lane) were separated with 10% SDS-PAGE and transferred onto PVDF membranes. Following blocking with non-fat milk at room temperature for 2 h, membranes were incubated with primary antibodies (all 1:1,000) as follows: OTX1 (cat. no. ab25985; Abcam), matrix metalloproteinase (MMP)2 (cat. no. ab181286; Abcam), MMP9 (cat. no. ab76003; Abcam), tissue inhibitor of MMP (TIMP)2 (cat. no. ab180630; Abcam), Wnt9 (cat. no. ER60346; Huabio), β-catenin (cat. no. ab223075, Abcam), adenomatous polyposis coli (APC; cat. no. ab239828; Abcam), Glycogen synthase kinase (GSK)-3β (cat. no. ab32391; Abcam) and Axis inhibition protein (AXIN)2 (cat. no. ab109307; Abcam) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated goat Anti-Rabbit IgG H&L secondary antibody (1:10,000; cat. no. ab205718, Abcam) for 1 h at room temperature. β-actin (1:1,000; cat. no. ab8226; Abcam) was used as an internal reference protein. The bands were visualized using a Novex^™ ECL Chemiluminescent Substrate Reagent kit (Thermo Fisher Scientific, Inc.). The grey value was analyzed using ImageJ software (Version 1.45s; National Institutes of Health).