E coli o111 b4

Biotin-conjugated lipid A (10 μg), LPS (50 μg), Pam3CSK4 (50 μg), and MDP (50 μg) from E. coli O111:B4 (InvivoGen) were immobilized onto 8 μl of streptavidin Sepharose beads (Thermo Fisher Scientific). For pull-down assay, the beads were washed three times in lysis buffer to remove unconjugated ligands and incubated with transfected cell lysates at 4°C for 12 hours. For the competition assay, 20 μg of unlabeled LPS, lipid A, Pam3CSK4, or MDP from E. coli O111:B4 (InvivoGen) was incubated with cell lysates for 60 min at room temperature before adding biotinylated LPS-conjugated streptavidin beads. The beads were washed in lysis buffer, and the precipitates were eluted in 1 × SDS loading buffer followed by immunoblotting analysis. To evaluate LPS binding to endogenous HyCaspA, 120 to 150 polyps were pretreated with 100 μM Z-VAD-FMK (Sigma-Aldrich) for 2 hours and lysed in the Hydra lysis buffer. For standard pull-down assay, 50 μg of biotin-conjugated LPS, lipid A, or MDP was immobilized onto streptavidin Sepharose beads followed by incubation with Hydra lysates. To examine LPS binding to purified recombinant HyCasps, 1 μg of Flag-purified HyCasp proteins (catalytic-cysteine mutants) was added to 250 μl of cell lysis buffer to perform pull-down assay.