Nanog

The general procedure for immunoblotting was described in a previously published report^{67 (link)}, except that cell lysates were prepared using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Carlsbad, CA) containing protease inhibitor and phosphatase inhibitor cocktails (EMD Millipore, Billerica, MA). The primary antibodies used in this study were purchased from Cell Signaling Technology (POU5F1; cat# 2840), EMD Millipore (NANOG; cat# MABD24) and MP Biomedicals (ACTIN; cat# 08691001). HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). The antibodies used in this study have been used or validated in separate work previously published by our and other groups.

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.