Production of Cells with Targeted Integration of Gene Variants of Human ABC Transporter for Stable and Regulated Expression Using the Flp Recombinase System - Protein Misfolding and Cellular Stress in Disease and Aging - page 147

protein levels were then normalized to the ratio of ABCG2
WT/GAPDH. Although mRNA levels were almost the same
in the WT and SNP variants (F208S and S441N), protein
levels of those variants were markedly decreased (Fig. 3d ).
A standard curve plotting protein amounts versus relative
scan intensity used to calculate the relative protein levels is
given in Fig. 4c .
4. To gain insight into the molecular nature of ABCG2 WT and
those two SNP variants, we performed immunoblot analysis
experiments with cell lysate samples under reduced or non-
reduced conditions. Figure 4a shows the effect of ME treat-
ments on the SDS-PAGE migration of ABCG2 WT, F208S,
and S441N proteins. When the cell lysate samples from
Flp-In-293 cells expressing those proteins were applied to
SDS-PAGE without ME treatments, one major band was
Fig. 4. Immunoblot detection of ABCG2 WT, F208S, and S441N proteins expressed in Flp-In-293 cells. ( a ) Effect of mer-
captoethanol (ME) on the status (homodimer or monomer) of ABCG2 WT and the SNP variants. Cell lysate samples (20 m g
of protein) were subjected to SDS-PAGE after treatments with or without ME; and, thereafter, ABCG2 WT, F208S, and
S441N proteins were detected by immunoblotting with the BXP-21 antibody, as described in Subheading 3.3.3. By ME
treatments, ABCG2 WT, F208S, and S441N proteins were changed from homodimers to monomers. ( b ) Effect of Endo H
or PNGase F treatments on the glycosylated status of ABCG2 WT and the SNP variants. Cell lysate samples (20 m g of
protein) were treated with Endo H or PNGase F, as described in Subheading 3.3.3. ABCG2 WT, F208S, and S441N proteins
in the resulting samples were analyzed by immunoblotting with the BXP-21 antibody. The apparent molecular weights of
mature and non-glycosylated ABCG2 WT were 81,000 and 72,000, respectively. ( c ) Schematic illustrations of N -linked
oligosaccharides. Arrowheads indicate the sites of Endo H- or PNGase F-cleavage. X and/or Y = AcNeu-Gal-GluNAc. Data
are from our previous publication ref. 24 with permission.