Biotinylated sna lectin

Methods for Western blotting were described in our previously published report16 (link). The primary antibodies used in this study were purchased from R&D Systems (ST6GAL1; cat# AF5924), Cell Signaling (POU5F1; cat# 2840), Millipore (NANOG; cat# MABD24) and MP Biomedicals (ACTIN; cat# 08691001). HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). For SNA lectin-mediated blotting, 10 ug of total proteins from each sample were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes with transferred proteins were blocked using a polyvinyl alcohol solution to prevent non-specific binding. After blocking, the membranes were reacted with PBS containing Triton X-100 (0.1%) and biotinylated SNA lectin (2 μg/ml; Vector Laboratories, Burlingame, CA) at 4 °C for 2 hours. After thorough washing, the membranes were reacted with HRP-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 30 minutes. After thorough washing, the chemiluminescence of an ECL substrate catalyzed by HRP was detected using film exposure.

Human pancreatic cancer cells were dissociated with trypsin, washed twice with HBSS containing 2% fetal bovine serum and 10 mM HEPES (staining buffer). Cells were stained and incubated on ice for 30 minutes with the following primary antibodies: ABCG2 (R&D Systems Inc, 1:50), FITC-conjugated CD24 (BD Biosciences, 1:100), APC-conjugated CD44 (BD Biosciences, 1:100), biotinylated SNA lectin (Vector Laboratories, 1:100), or MALII lectin (Vector Laboratories, 1:100). After stained with appropriated secondary antibody, the stained cells were analyzed by FACSCalibur (BD Biosciences).