Protein Extraction and Western Blot Analysis

The throat tissues were mechanically grinded in a glass homogenizer, the RIPA lysis buffer (Beyotime, Jiangsu, China) and protein phosphatase inhibitor (Beyotime, Jiangsu, China) were added to lysed the samples for 1 h on ice. In order to acquire supernatant, homogenates were centrifuged for 10 min at 12,000 rpm and 4 °C [29 (link)]. BCA protein test kit (Beyotime, Jiangsu, China) was used for the protein quantification. Then boiling the soluble extracts with Loading buffer (Beyotime, Jiangsu, China) for 10 min. The equal amount of protein exact of samples were electrophoresed on a 10% SDS-PAGE, then the electrophoretic proteins are transferred onto a PVDF. After blocking, membrane was probed with goat polyclonal antibodies against NF-κB p65 (1:2000; Proteintech, USA), COX-2 (1:1000; Proteintech, USA), GAPDH (1:5000; Proteintech, USA). The second day, rinsing the membrane with tris-buffered saline Tween (TBST) 3 times for 10 min, and incubating with goat anti-rabbit IgG (1:5000; Proteintech, USA), which can conjugate with appropriate secondary antibody HRP. Afterwards, cleaning the membrane again with TBST and treated with a chemiluminescence analysis kits (Beyotime, Jiangsu, China) for visualization. The bands were analyzed (relative to GAPDH expression) by using Gel-pro analyzer [30 (link)].