were harvested by centrifugation and washed once in 20 mM Tris-HCl, pH 8.0, before the cell pellet was frozen and stored until use. The frozen cell pellet was resuspended in 200 mL of 20 mM Tris-HCl, pH 8.0, and cells disrupted using a probe sonicator before collecting the inclusion bodies by centrifugation. Inclusion bodies were washed 4 times PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674025 with 20 mM Tris-HCl, pH 8.0, and then once with 1% Triton X-100 in 20 mM Tris-HCl, pH 8.0. The remaining pellet was washed four more times with 20 mM TrisHCl, pH 8.0, before solubilization in 25 mL of 20 mM Tris-HCl, pH 8.0, 6 M guanidinium hydrochloride, 10 mM dithiothreitol. The CVT-3146 solubilized material was centrifuged for 30 min at 12,0006g and the supernatant diluted drop by drop into 4 liters of 20 mM Tris-HCl, pH 9.3, 0.3 M arginine monohydrochloride, 1 mM EDTA, 0.2 mM GSSG, and 1 mM GSH, pH 9.3. After 48 h at 4uC, under PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19675955 agitation, the liters were filtered, and concentrated by ultrafiltration using a 10 kDa cut-off prep/scale TFF cartridge to approximately 300 mL. After centrifugation for 30 min at 12,0006g, the supernatant was concentrated in a 10 kDa membrane using a Amicon ultrafiltration device until a final volume of 30 mL. Samples were centrifuged again, and then purified by HPLC as below. SDS-PAGE and Edman degradation Samples were treated with 46 NuPAGE lithium dodecyl sulfate sample buffer and 106 sample reducing reagent, then loaded in NuPAGE-Bis-Tris 4% to 12% gels with 2- ethanesulfonic acid running buffer. Gels were Coomassie blue stained. Labeling of Agalectin Agalectin was labeled with FITC using the Fluoreporter FITC protein labeling kit from Invitrogen/Molecular Probes, as per manufacturer’s instructions. Glycan Microarrays The Glycan-protein interaction resource was developed by the Consortium for Functional Glycomics under the directions of Drs. David F. Smith, and Jamie Heimburg-Molinaro. Experiments were performed under the request protocol_3072. Agalectin was labeled with FITC as described by the manufacturer, and dialysed against binding buffer: 20 mM Tris, 150 mM NaCl, 2 mM Ca2+, 2 mM Mg2+. Then, 0.05% tween-20, and 1% BSA were added to the sample. Experiments were performed as described in detail at: http://www.functionalglycomics.org/static/consortium/ consortium.shtml. Fractionation of An. gambiae SGH One hundred pairs of female An. gambiae SGH were dissected under the microscope, sonicated extensively, and centrifuged for 10 min at 13,0006g. The supernatant was collected and loaded in a Superdex G-75 10/300 GL gel-filtration column equilibrated in PBS pH 7.4 containing 0.1% PEG 4000. Elution was carried out at 0.5 ml/min, with Mosquito Salivary Lectin 0.5 ml fractions. Addition of PEG was critical in order to recover the hemagglutinating activity of the SGH. The fractions were subsequently tested for hemagglutinating activity as above. Control experiments demonstrated that the presence of PEG 0.1% did not affect the lectin 
properties of the SGH. In order to estimate the correct molecular weight of the candidates, the column was calibrated with Conalbumin, BSA, Ovalbumin, and Approtinin. bands, followed by tryptic digestion and nano-LC MS/MS essentially as described. The MS results were matched against a database of Anopheles gambiae salivary gland transcriptome. HL-60 cells culture and adhesion to P-selectin P-selectin was immobilized overnight in Microfluor 2 Black “U”bottom 96 well microplate, followed by washing 3 times in PBS. Blockade of non-specific binding si