Hrp labeled goat anti mouse igg

Carassius auratus gibelio caudal fin cells were collected and lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4; Beyotime, China). The extracted cellular proteins were separated in 10% SDS-PAGE gels using a Mini-PROTEAN^® 3 Cell (Bio-Rid) system, and then transferred onto a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot^® SD Semi-Dry Transfer Cell system (Bio-Rad, USA). The membranes were subsequently blocked with phosphate buffered solution (PBS) containing 5% (w/v) non-fat milk at room temperature (RT) for 2 h and then incubated overnight at 4°C with primary antibodies (1:1,000 dilution). Primary antibodies include: mouse polyclonal antibodies ORF23 and ORF141 were prepared by our laboratory (antibody potency analyses can be seen in Supplementary Figures 1, 2, and information on pertinent recombinant plasmids and primers can be found in Supplementary Table 2). After washed with PBST, the cells were incubated with HRP-labeled Goat Anti-Mouse IgG as the secondary antibody (Abbkine, China, 1:5,000 dilution) for 2 h at RT, and then washed with PBST. The proteins were visualized by chemiluminescent method using the ECL and detected using ChemiDoc™ Imaging System (Bio-Rad, USA).

TSD was extracted by the research group, and the total saponin content was 61.7% [17 ]. Other reagents were colchicine (purity rate >99%) (Meilunbio Co., Ltd., J1225AS), colchicine tablets (Xishuangbanna Pharmaceutical Co., Ltd.), MSU (Shanghai Yuanye Biotechnology Co., Ltd., S24J9I64369), rat IL-1β ELISA kits, rat TNF-α ELISA kits (Wuhan Boster Bios Co., Lot : EK0393 and EK0526, respectively), TLR4 antibody, MyD88 antibody, NF-κB antibody, β-actin antibody (Abcam, ab13556, ab2064, ab16502, and ab8227, respectively), Lamin B antibody (Affinity, AF5161), PCNA antibody (Abbkine, A01040), GAPDH antibody (Servicebio, GB12002), HRP-labeled goat antirabbit IgG, HRP-labeled goat antimouse IgG (Abbkine A21020 and A21010), and ECL chemiluminescence kit (Thermo 32109).