Glycan Characterization of Cell Lines

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.