Cy5 labeled

Cell pellets from the blood samples collected at different time points during SAL-1 or AMRU-1 inoculations were stored in TRIzol. RNA was extracted and processed to be run in a customized microarray assay detecting both core and subtelomeric genes. The previously described microarray hybridization protocol was used for this study, with several modifications (109 (link)). In brief, 100 ng of cDNA was used for the subsequent 10 rounds of amplification to generate aminoallyl-coupled cDNA for the hybridizations as described previously (109 (link)). 17 μl (∼5 μg) of each Cy5-labeled (GE Healthcare) cDNA of the sample and an equal amount of Cy3-labeled (GE Healthcare) cDNA of the reference pool were then hybridized together on a customized microarray chip using a commercially available hybridization platform (Agilent) for 20 h at 70°C with gentle rotation. Microarrays were washed and immediately scanned using Power Scanner (Tecan) at 10-μm resolution and with automated photomultiplier tubes gain adjustments to balance the signal intensities between both channels. The reference pool used for microarray was a mixture of 3D7 parasite strain RNA collected every 6 h during 48 h of the full IDC.

In order to further confirm the different abundance of significant glycans, the SDS-PAGE and lectin blotting analysis was performed with 3 lectins (HHL, AAL, and Jacalin)in pooled saliva group samples and 4 lectins (HHL, PNA, PHA-E+L, and AAL) in both pooled saliva group samples and pooled serum group samples of the normal control, the untreated KBD, and the treated KBD groups separately. In brief, the samples were run on a 10% SDS-PAGE polyacrylamide resolving gel and a 3% stacking gel. Afterwards, the proteins in the gels were transferred to a PVDF membrane (MilliporeSigma, Burlington, MA, USA) with a wet transfer unit (Hoefer Scientific, Holliston, MA, USA) at 100 V for 1.5 h. The membrane was washed twice with TTBS (10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween-20, pH 7.5) and blocked with Carbo-Free Blocking Solution (Vector, Burlingame, CA, USA) for 1 h at room temperature. The membrane was incubated with Cy5-labeled (GE Healthcare, Buckinghamshire, UK) lectins (2 mg/) at 4 °C overnight in the dark. Finally, the membrane was scanned using a phosphorimager (Molecular Dynamics Inc., Sunnyvale, CA, USA) [9].