2.6. Lectin blot analysis

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].