Agarose

In vitro encapsulation assay was performed according to previous report51 (link). Briefly, Ni-NTA agarose beads (Qiagen) were equilibrated in TBS containing 5 mM CaCl₂, and then incubated with renatured His-tagged rCfLec-3, rCRD1, rCRD2, rCRD3, mutated CRDs and rTrx in a 1.5 mL tube with shaking at 4 °C overnight, respectively. A 48-well cell culture plate (Costar) was coated with 1% agarose (Qiagen). The diluted hemocytes (about 10⁶ Cells mL⁻¹) in Leibovitz L-15 medium (Sigma) were added to each well of the agarose-coated plate. After the hemocytes were allowed to settle down for at least 10 min, 1 μL (100-120 beads) of the protein-coated agarose beads was added to each well, and the plate was incubated at 18 °C for 6 h. The encapsulation of the agarose beads was observed under a light microscope. For each recombinant protein, the assay was performed in three different wells for statistical analysis. To test whether in vitro encapsulation was specifically incited by CfLec-3, 5 μL protein-coated beads were placed in a microcentrifuge tube, and 5 μg of rCfLec-3 antibody (in TBS, pH 7.5) was added and incubated at 4 °C overnight with shaking. Then the beads were washed with TBS and suspended in 5 μL TBS. In vitro encapsulation assay was performed as described above.

N-Terminal histidine tagged D154AFNR and CRP, His₆-D154AFNR, and His₆-CRP were expressed in and purified from E. coli Rosetta™(DE3) (Novagen/EMD Bioscience, Philadelphia, PA), respectively. We supplied 100 μM cAMP into the buffer for purification of His₆-CRP. Bacteria containing recombinant plasmid were grown at 37°C to an OD₆₀₀ of 0.4 in LB, 0.5 mM IPTG was then added, and culture growth was continued for 3 h. Cells were harvested and stored at −80°C overnight. The cell pellet was suspended in lysis buffer (20 mM Tris [pH 7.9], 500 mM NaCl and 10% glycerol) and lysed by sonication. The lysate was centrifuged and the resulting supernatant was mixed with Ni-NTA agarose (Qiagen, Valencia, CA) for 1 h. The agarose was washed with 50 mM imidazol twice and then protein was eluted with 200 mM imidazol. The protein was >95% pure as estimated by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). The protein was diluted into gel-shift/footprinting assay buffer (20 mM Tris [pH 7.5], 50 mM KCl, 1 mM dithiothreitol, 10% glycerol and 200 μM cAMP) to a concentration of 4 pmol/μl, and stored at 4°C.