Horseradish peroxidase labeled goat anti rabbit secondary antibody

Cells were washed twice in ice-cold PBS, and then solubilized in RIPA lysis buffer (Vazyme). Proteins extracted were quantified using a Bicinchoninic Acid Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Equal amounts of each protein sample (30 µg/lane) were subjected to 10% SDS-PAGE and were transferred to polyvinylidene fluoride membranes, which were blocked with 5% skimmed milk at room temperature for 1 h. Members were incubated overnight at 4°C with antibodies targeting Hrd1 (cat. no. ab170901; 1:2,000; Abcam), Nrf2 (cat. no. sc-13032; 1:2,000; Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc.), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721; 1:3,000; Abcam) for 1.5 h at room temperature. β-actin was used as a loading control. Protein bands were visualized by enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore). ImageJ 1.45 software (National Institutes of Health) was used to perform densitometric analysis of each band.

Total proteins were extracted from cells using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins were quantified using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.), followed by western blot analysis. 40 µg proteins from cell lysates were subjected to 12% SDS-PAGE. Once proteins were transferred to nitrocellulose membranes, they were incubated with antibodies targeting β-actin (cat. no. 58169, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CCR8 (cat. no. ab32131, 1:500; Abcam, Cambridge, UK), E-cadherin (cat. no. 3195, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), matrix metalloproteinase (MMP)-2 (cat. no. 40994, 1:1,000; Cell Signaling Technology, Inc.) and vascular endothelial growth factor (VEGF)-C (cat. no. 2445, 1:1,000; Cell Signaling Technology, Inc.), followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. ab6721, 1:3,000; Abcam) for 1.5 h at room temperature. Protein bands were visualized by Millipore enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore, Billerica, MA, USA). ImageJ 1.45 software (NIH) was used to perform densitometric analysis of each band.