Enhanced chemiluminescence substrate

Pancreatic samples were snap-frozen and homogenized in RIPA buffer (Sangon Biotech, Shanghai, China) on ice. After centrifugation at 12000 rpm for 10 min at 4°C, the protein content of the supernatant was mixed with loading buffer. An equal amount of protein per sample was resolved on gradient gels (15% Tris-glycine gels) and electroblotted on PVDF membranes, which were then incubated with primary antibodies (rabbit polyclonal anti-H3cit, Abcam; rabbit polyclonal anti-GAPDH, Servicebo) at 4°C overnight and subsequently with appropriate HRP-conjugated secondary antibodies (Servicebo, Wuhan, China) for 1 h at room temperature. The blots were developed with enhanced chemiluminescence substrate (Sangon Biotech, Shanghai, China). Blots were quantified using ImageJ software.

According to a previous report, CFA infection increases the expression of Nav1.7 [49 (link)]. Nav1.7 can be upregulated in L4/5 DRG neurons in a certain evoking situation [50 (link)]. Therefore, L4-5 DRG samples from different groups were obtained. Protein was isolated using a plasma membrane protein isolation kit (Cat. No. ab65400, Abcam Trading (Shanghai) Company Ltd., Shanghai, China). Rabbit anti-mouse monoclonal Nav1.7 antibody (Cat. No. 62758, dilution 1:5000, Abcam Trading (Shanghai) Company Ltd., Shanghai, China) was used as the first antibody. Polyclonal Goat Anti-Rabbit IgG H&L (Cat. No. ab6721, dilution 1:3000, Abcam Trading (Shanghai) Company Ltd., Shanghai, China) was used as a secondary antibody. A rabbit anti-mouse β-actin polyclonal antibody (1:2000 dilution; Cat. No. 4967, Cell Signaling Technology, Danvers, MA, USA) was used as a loading control. All protein bands were visualized by using an enhanced chemiluminescence substrate (Sangon Biotech, Co., Ltd., Shanghai, China). The image intensity of protein bands was quantified by using NIH ImageJ software (Bethesda, MD, USA).