2.11 3D structure prediction
The Galectin-3 structure of the mouse used in this study was obtained from the PDB database with the accession number 7CXB, while the mouse TLR4 protein had the accession number 2Z64. Initially, protonation processing under neutral conditions (pH = 7) was performed using the H++3 online server. Subsequently, UCSF Chimera software was utilized to eliminate heteroatoms and water molecules from the crystal structure, retaining only the protein structure with Amber14SB charge allocation. Next, the professional protein-protein docking tool HDOCK is utilized for molecular docking, while the experience-based iterative scoring function ITScorePP is employed for both molecular docking and configuration scoring. A negative score indicates successful binding of molecules, with greater absolute values indicating stronger binding ability. The maximum number of output configurations for docking is set at 100, with only the top 10 configurations being scored and analyzed using Confidence scores to determine reliability. If the value exceeds 0.7, it signifies a reliable docking score and a high likelihood of molecular binding. Conversely, if the value falls below 0.7, it indicates a low reliability in the docking score and a diminished possibility of binding. Choose the docking configuration with the highest docking score and confidence score for subsequent analysis. Utilize PyMOL2.04 for 3D mapping analysis, and employ academic Maestro for 2D interaction analysis, statistical determination of interaction type, distance, and quantity.
2.12 CO- immunoprecipitation (CO-IP)
Revised sentence: Co-immunoprecipitation (Co-IP) assays were conducted in accordance with established protocols43. The cells were directly scraped off using a cell scraper in immunoprecipitation lysis buffer NP-40 (Beyotime China) containing phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor. After being lysed for 30 minutes on ice, the cells were centrifuged at 12,000 rpm for 30 minutes at 4℃. The resulting supernatant was collected and subsequently incubated with primary antibodies (TLR4 and Gal-3) or isotype immunoglobulin G (IgG). (#3900, Cell Signaling Technology, Shanghai, P.R. China), gentle rocking 2 h at 4°C. Subsequently, 35 µL of protein A/G beads (#sc‐2003, Santa Cruz, Shanghai, China) were added to each immunoprecipitation mixture and allowed to stand overnight at 4°C. On the following day, the mixtures were subjected to five rounds of cold 1×Co-IP buffer washes, followed by denaturation of bound protein with 1× sample buffer. The resulting supernatants were collected and utilized for SDS-PAGE Western blot analysis.