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 146

Fig. 3. Schematic illustration of human ABCG2 as well as the expression of ABCG2 WT, F208S, and S441N in Flp-In-293
cells at the mRNA and protein levels. ( a ) Arrows indicate the positions of amino acid substitutions (Phe208Ser and
Ser441Asn) in the non-synonymous SNP variants of F208S and S441N. Cysteine residues, that is, Cys592, Cys603, and
Cys608, are also indicated by arrows . Cys592 and Cys608 in the extracellular loop form an intra-molecular disulfide
bond, whereas Cys603 is involved in homodimer formation via an inter-molecular disulfide bond. N -linked glycosylation
reportedly occurs at Asn596 (N596). The sphere labeled ABC indicates the ATP-binding fold. ( b ) The mRNA level was
analyzed by RT-PCR with total RNA extracted from Flp-In-293 cells expressing ABCG2 WT, F208S, or S441N. ( c ) The
relationship between the relative intensity of ABCG2 immunoreactivity and the amount of cellular proteins. ( d ) For com-
parison of the protein levels, the cell lysate of each cell population was analyzed by immunoblotting with the ABCG2-
specific monoclonal antibody (BXP-21) or the GAPDH-specific antibody after PNGase F treatment. The signal intensity
ratio (ABCG2/GAPDH) was normalized to the WT level. Data are from our previous publication ref. 24 with permission.
F208S, and S441N were individually expressed in Flp-In-293
cells by using the Flp recombinase system. As shown in
Fig. 3b , mRNA levels of ABCG2 WT as well as F208S and
S441N were evenly represented in Flp-In-293 cells, where
the mRNA levels of ABCG2 and GAPDH were measured by
RT-PCR. On the other hand, ABCG2 WT, F208S, and
S441N as well as GAPDH proteins were detected by immu-
noblotting, and their expression levels were quantified. For
this purpose, we treated all of the samples with PNGase F and
ME to remove glycomoieties and to break the cysteinyl disul-
fide bond forming a homodimer. Since there was a linear rela-
tionship between the signal intensity of immunoblotting and
the logarithmic value of the amount of ABCG2 protein
applied to the electrophoresis, the expression level of ABCG2
or GAPDH in cell lysate samples could be quantitatively esti-
mated based on the linear relationship. The relative values of