Western Blotting and Glycosidase Digestions

Protein from the untreated cells (0 h) as well as from those subjected to 100 μM biotin for the indicated times was extracted by the addition of TE buffer (0.1 M TRIS-PO₄, 4% SDS, 4 mM Na-EDTA, 40% glycerol, 0.04% bromophenol blue, and 0.04% β-mercaptoethanol; materials from Sigma-Aldrich), and then the samples were sonicated. In certain experiments, when indicated, the cells were pre-treated with 1 mM 4-PBA, 2 μm MG132, or 10 nM BAF overnight prior to biotin addition. Equal amounts of the protein samples were loaded onto 7.5% polyacrylamide gels. For glycosidase treatments, cell lysates containing 20 μg of protein were subjected to 20 U Endoglycosidase H (Endo H) (Sigma, cat.11088726001) or 20 U PNGase F (Roche, cat.11365185001) according to the manufacturer’s protocols. Blots were probed with anti-ABCG2 (Bxp-21, Abcam, cat. ab3380) or anti-β-actin (Sigma, cat. A1978) primary antibodies, and subsequently developed with HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (Abcam, cat. ab97023). Detection was performed by luminography using Clarity Western ECL Substrate (Bio-Rad, cat. 1705060). For quantification, densitometry was carried out by the ImageJ software.