Lipid a

Recombinant FL-PXDN, PXDN 29-250aa or PXDN 251-609aa was added into 100 μL PBS containing 4 x 10⁸ live P. aeruginosa strain K or E. coli K12. The mixture was incubated at RT for 1 h. Bacteria were spun down at 3099 x g for 5 min. The cells were washed twice by 5 x initiating volume of PBS. Bacteria were lysed in 2 x SDS-PAGE loading buffer and the lysates were subject to immunoblot analysis using anti-His antibody. Protein bands were visualized by chemiluminescence. Negative controls contained only PXDN or bacteria. Quantitative analysis was carried out by using ImageJ software (The National Institute of Health). In some experiments, LPS-deficient E. coli stains were utilized. LPS-deficient E. coli strains and their parent strain K12 BW25113 were from The Coli Genetic Stock Center at Yale University. In bacteria and plasma PXDN binding assay, 4 x 10⁸E. coli or P. aeruginosa in 50 μL PBS were mixed with 50 μl of human plasma. Control groups were cells or plasma alone. In some experiments, lipid A (Sigma-Aldrich Cat. #L5399) was used. In brief, indicated amount of lipid A was mixed with 150 nM of recombinant PXDN in 600 μL PBS. The mixture was shacked at RT for 15 min. Then 2 x 10⁸ live E.coli K12 cells were added into the mixture and incubated at RT for additional 30 min. Bacterial pellets were obtained and subject to immunoblot analysis.

As reported^{34 (link),35 (link)}, lipid-A (sigma) were diluted in DMSO an initial amount of 50 μM/well, and the solvent (80 μL) was coated in Nunc-Immuno™ MicroWell™ 96-Well Plates (Thermo Scientific) by overnight incubation at 4 °C. Remove the solvent and blocked with a 3% (w/v) BSA in PBS for overnight at 4 °C. Thoroughly washed with PBS containing 0.05% Tween-20, and subsequently incubated with twofold serial dilutions (starting at 100 μM) of His-tagged proteins (MucA^peri, MucB, and MucB mutants) for overnight at 4 °C, the concentration gradients are 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100 μM. The plates were then washed for five times with PBS, incubated with mouse anti-His antibody (Invitrogen) for 2 h, washed again for five times with PBS, incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody diluted 1:5000 in 1% BSA in PBS, and washed, as a final washing stage, for five times again with PBS. Finally, the bound proteins were detected using TMB-ELISA substrate solution (Horseradish Peroxidase-HRP, beyotime). After incubated for 30 min at room temperature, the reaction was stopped by addition of 1 N HCl. Optical density (OD) was subsequently measured at 450 nm. Each experiment was performed three times, and each point is a mean of three replicates ± SD.