Mal 2

Lectin blot analyses were conducted using biotinylated peanut agglutinin (PNA, Vector Laboratories, Burlington, ON, Canada), specific to carbohydrate sequence Gal-β (1–3)-GalNAc, and biotinylated Maackia amurensis lectin II (MAL-II, Vector Laboratories), specific for α2–3-linked sialic acid. Twenty-five micrograms of protein from each rat brain homogenate was resolved on an 8% SDS-PAGE gel and transferred to nitrocellulose membranes. Blots were blocked with 3% bovine serum albumin and then incubated overnight at 4 °C with biotinylated lectins (see Table 1 for dilutions used). Subsequently, membranes were incubated with streptavidin-horseradish peroxidase conjugate (1:1000; GE Healthcare Life Sciences, Baie-d’Urfé, QC, Canada). Lectin reactivity was detected by enhanced chemiluminescence (ECL) using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., Rockford, USA). The total intensities of the stained protein bands were quantified by ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.net/, 1997–2016) and normalized for the combined intensity of protein bands on the same lane stained with Ponceau Red.

Cells fixed with 4% paraformaldehyde, and permeabilized at room temperature in 0.1% Triton X-100 for 30 minutes. After blocking with blocking buffer (Roche), the cells were incubated with primary antibody overnight at 4°C and with secondary antibody for 2 hours at room temperature. The primary antibodies were used at the following dilutions: anti-ST6Gal1 (ST6Gal1-M2, IBL, 1:50), SNA (Vector Laboratories, 1:100), MALII (Vector Laboratories, 1:100), and ABCG2 (R&D, 1:50). Cells were counter-stained with Hoechst dye (Sigma-Aldrich) to visualize the cell nuclei. Immunostaining images were obtained using a fluorescence microscope (Leica Microsystems Inc).

Proteins (50 μg) were subjected to SDS-PAGE in a 10% acrylamide gel, and the resulting bands were transferred onto nitrocellulose membranes. The membrane was blocked by incubation with [0.5% BSA, 0.15 M NaCl, 0.1 mM CaCl₂, 0.01 mM MnCl₂.4H₂O, 0.01 M HEPES.Na⁺ (pH 7.5)] (HEPES-BSA) overnight at 4 °C, and plant lectin-binding activity was detected by incubating the membrane with HEPES-BSA containing biotinylated ConA, SBA, PNA, SNA, MAL-II, RCA-I, jacalin, UEA-I, DBA, and WGA plant lectins (0.2 μg/ml; Vector Laboratories, Inc., Burlingame, CA, USA) (See Table 2 for binding specificities) for 1 h at room temperature. The membrane was then washed three times, for 15 min each, with HEPES-BSA. Bound biotinylated lectin was detected by incubating the membranes with peroxidase-conjugated streptavidin (0.5 μg/ml) for 30 min at room temperature. The membranes were washed three times, for 15 min each, with HEPES-BSA, and bound peroxidase activity was detected by incubation with 3,3′-diaminobenzidine in the presence of CoCl₂ and H₂O₂⁶¹ .