β actin

Total cell proteins were prepared using IP lysate (P0013, Beyotime, China) mixed with protease and phosphatase inhibitors (HY-K0010 and HY-K0023, MCE, China). Cell lysates were centrifugated at 4℃ and their concentrations were determined using a BCA Protein Assay Kit (Pierce Biotechnology, USA). Next, the samples were subjected to separation through electrophoresis with 10–12% Tris SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with 5% skimmed milk at room temperature and primary antibodies at 4℃ overnight. Next, secondary horseradish peroxidase (HRP) antibodies were applied at room temperature. Finally, proteins were visualized using ECL (WB012, Share-Bio, Shanghai) detection regent and a Bio-Rad system. The antibodies used were directed against GFRA1 (ab84106, Abcam, 1:1000), GDNF (ab18956, Abcam, 1:1000), RET (14,556, Cell Signaling Technology, 1:1000), β-actin (30101ES50, Yeasen, China, 1:5000), mTOR (2983, CST, 1:1000), p-mTOR (5536, CST, 1:1000), S6K (2708, CST, 1:1000), p-S6K (9204, CST, 1:1000), BECN1 (3495, CST, 1:1000), LC3 (12,741, CST, 1:1000), P62 (8025, CST, 1:1000), cleaved caspase-3 (9661, CST, 1:1000) and its corresponding HRP-conjugated antibodies (anti-mouse, 115–035-003 and anti-rabbit, 111–035-003, Jackson ImmunoResearch, 1:10,000 for both).

The pathogenic strain C48-1 (A:1) of P. multocida was isolated from chicken with fowl cholera in Jiangsu, China, in 1953 and was stored at our laboratory. The murine peritoneal exudate macrophages were isolated from C57BL/6N mice. The DF-1 and HD11 cells were provided by our laboratory. The antibodies used were as follows: antibodies against ERK1/2, p38, JNK, p65, p-ERK1/2, p-p38, p-JNK, and p-p65 (Cell Signaling Technology, USA); procaspase-1 and IL-1β (Abcam, England); GSDMD or GSDMD(-N), ASC, and caspase-1 p20 (Affinity, USA); NLRP3 (Proteintech, China); TLR1, TLR2, and TLR4 and IgG isotype-matched control antibodies (Proteintech, China); and β-actin (Yeasen, China). The NF-κB (BAY11-7082), JNK (SP600125), p38MAPK (SB203580), and ERK (U0126) inhibitors were purchased from Sigma-Aldrich (USA). The IRDye 800CW-conjugated anti-rabbit IgG or anti-mouse IgG antibodies were purchased from Li-Cor Biotechnology (USA). In this study, 5- to 8-week-old C57BL/6N mice were purchased from Liaoning Changsheng Biotechnology (Liaoning, China), and SPF chicken serum was provided by the National Engineering Research Center of Veterinary Biologics Corp. (Harbin, China).

Cells were washed and lysed with RIPA buffer (WB3100, NCM, China) containing protease inhibitors cocktail (B14001, bimake) on ice for 10 min. Then, protein lysate followed centrifugation in 4 °C for 10 min and the supernatant was collected. Protein supernatant were prepared with 5 × SDS loading buffer (P1040, Solarbio) and denatured at 100 °C for 5 min. Appropriate protein of samples were separated by 4–20% Genshare PAGE gel electrophoresis and electroblotted into NC membranes on eBlot™ L1 Protein Transfer System (GenScript). The membranes were incubated in 5% non-fat powdered milk (Cat No. 36101; Yeasen, Shanghai, China) in TBST (TBS with 0.1% Tween20) for 1 h at room temperature, followed by incubation with primary antibodies against specific proteins overnight: β-actin (1:5,000, Yeasen, 30101ES50), HSP90 (1:1000, 13171-1-AP, Proteintech), and OPA1(1:1000, Abcam, ab42364). The primary antibodies were diluted in universal antibody diluent (WB500D, NCM, China). The membranes were washed thrice of 10 min each time and incubated with the HPR-conjugated goat anti-mouse (1:10,000, Jackson ImmunoResearch, 115-035-003) or rabbit secondary antibodies (1:10,000 Jackson ImmunoResearch, 111-035-003) for 1 h at room temperature. Enhanced chemiluminiscence (ECL) was performed using the ECL kit (WB012, share-bio, China), visualized by the Bio-Rad system.