Figure 3.

Podocalyxin is a glycoprotein ligand of rBC2LCN. Dynabeads M-280 streptavidin (10 μl) immobilized with biotinylated anti-podocalyxin pAb (1 μg) was incubated with hydrophilic fractions of cells at 4°C overnight with agitation. After washing the beads with 200 μl of PBST three times, bound podocalyxin was eluted with 50 μl of TBS containing 0.2% SDS at 95°C for 10 minutes. Coeluted biotinylated anti-podocalyxin pAb was partially depleted with Dynabeads M-280 streptavidin (15 μl) at room temperature for 1 hour with agitation. The eluted fractions were run on 5%–20% acrylamide gel under reducing condition and transferred to polyvinylidene difluoride membrane. The membrane was stained with either HRP-conjugated rBC2LCN (1 μg/ml, top panel) or goat anti-podocalyxin pAb (0.1 μg/ml) followed by HRP-conjugated donkey anti-goat IgG (×10,000, bottom panel). The staining was performed in different membranes. Both 55- and 10-kDa bands are nonspecific, since both bands could be detected in the control immunoprecipitated samples. The 55-kDa band might be the heavy chain of goat anti-podocalyxin pAb used for the immunoprecipitation of podocalyxin, whereas the identity of the 10-kDa band is unknown. Abbreviations: ES, embryonic stem; HRP, horseradish peroxidase; iPS, induced pluripotent stem; MEF, mouse embryonic fibroblasts; pAb, polyclonal antibody.