Mal 1

MDCK, SIAT1, and hCK cells (Passage 3 and 23) were lysed with RIPA buffer, and lysates were treated with PNGase F and neuraminidases as described previously^{13 (link)}. Total protein concentration was determined by Pierce™ BCA assay kit (Thermo Scientific). Samples (30 μg/lane), plus the control of PNGaseF/neuraminidases mixture (Enzyme mix) loaded on SDS-PAGE gels were stained with Colloidal CBB (Bio-Rad), or transferred onto PVDF (EMD Millipore, wet-transfer system, 40 V for 2 h). The membranes were blocked with 5% (w/vol) BSA in TBST for 1 h at room temperature, and incubated with biotinylated lectins ConA (0.1 μg/ml), SNA (2 μg/ml), MAL-I (5 μg/ml), PHA-E (2 μg/ml) (Vector Laboratories), or O6 (1 μg/ml, anti-3-O-SGal antibody; mouse IgG-Fc) in TBST overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP-labeled streptavidin (Vector Laboratories), or goat anti-mouse IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc.) at 1:5,000 dilution for 1 h at room temperature, and the signals were analyzed on an Amersham™ Imager 600 (GE Healthcare Life Sciences) using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific). See Supplementary Figs. S1 and S2 for original, uncut blots and gels.

Indicated cell lines were seeded on glass coverslips coated with poly-lysine solution and incubated overnight at 37 °C. Cells were then fixed with 4% formaldehyde in PBS for 15 min at room temperature and washed with PBS. Samples were stained with 20 µg/mL WGA (Vector Laboratories, FL-1021-5), MAL I (Vector Laboratories, FL-1311), SNA (Vector Laboratories, FL-1301), or GNL (Vector Laboratories, FL-1241) fluorescent lectins in PBS for 1 h at room temperature. For staining of HNK-1/GlcA, cells were blocked for 1 h at room temperature with PBS + 5% BSA, then incubated with anti-HNK-1 primary antibody (Sigma C6680, 1:250) overnight at 4 °C in PBS + 0.5% BSA. Cells were washed and incubated with anti-mouse Alexa Fluor 488 antibody for 1 h at room temperature. All samples were washed and stained with Hoechst 33342 (Thermo H3570) to visualize nuclei, then mounted using Prolong Diamond Antifade (Thermo P36965).

For measuring the level of 2,3-linked sialic acid on the cell surface, cells were detached from plates using trypsin and stained with fluorescein-labeled MALI (Vector Laboratories). As a control, HeLa cells were treated with 100 milliunits/ml neuraminidase from Clostridium perfringens (Sigma-Aldrich) as described previously (10 (link)). Flow cytometry was performed using a BD Accuri flow cytometer (BD Biosciences), and the data were analyzed using the FlowJo program.