EXPERIMENTAL APPROACHES TO THE STUDY OF DRUG TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 549

experimental systems, site-directed mutagenesis of the transcription factor recogni-
tion sites can also provide useful confirmation.

Posttranscriptional Regulation Posttranscriptional regulation of transport proteins
may involve kinations, interaction with regulation factors, modification, and so on,
which may result in changes of specificity and localization. Here, we focus on inter-
actions with regulation factors. To investigate protein-protein interactions between
the transporter and regulatory factors and to identify putative regulation factors,
immunoprecipitation, pull-down assay, and the yeast two-hybrid method are often
employed. 63 - 65

Immunoprecipitation Immunoprecipitation is initiated by mixing lysates of
transporter- and the tag sequence-fused putative regulatory factor-expressing cells,
or putative regulation protein-expressing cells and membrane fraction prepared from
tissues. Here, we describe the use of transporter and putative regulatory protein-
expressing cells. Cultured cells stably expressing the putative regulatory protein and
cells transiently expressing the transporter are washed twice with phosphate-buffered
saline (PBS) and collected with a rubber policeman followed by centrifugation. The
pellets obtained are solubilized in RIPA-Y buffer. The cell lysates are mixed and then
incubated with the antitag sequence antibody prebound to Protein G Sepharose at
4 ◦ C for 4 hours, followed by centrifugation and washing three times with ice-cold
PBS. Samples are analyzed by Western blot analysis. If the construct of the putative
regulatory protein is fused to c-myc, anti-c-myc antibody is prebound to Protein G
Sepharose, and Western blot analysis is performed with antitransporter antibody. A
specific signal of the transporter on the blotting membrane suggests the occurrence
of a protein-protein interaction. 64

In general, to clarify interactions among proteins,

a single approach is not sufficient.

Biotinylation Study of Surface Proteins To investigate transporter expression
changes and localization, biotinylation is often performed. Although Western blot
analysis of the membrane fraction indicates the amounts of transporters in the cell
membrane including organelle membranes, a biotinylation study allows us to deter-
mine the amount of functional transporter localized on the plasma membrane.
At 48 hours after transfection of fluorescent protein-fused transporter, the cells
are harvested and washed three times with PBS. Cells (2.5 10 7 cells/mL) are then

incubated at 4 ◦ C with 0.5 mg/mL sulfo-NHS-LC-Biotin for 1 hour, washed with
PBS, incubated again with sulfo-NHSLC-biotin in the same manner, washed, and
solubilized in 20 mM phosphate buffer. The solubilized fraction is incubated at 4 ◦ C
for 30 minutes with 50

×

L of immobilized streptavidin, which is then washed three
times with PBS containing 0.02% Tween 20 and subjected to Western blot analysis. 65
To study interactions and change of localization under physiological conditions,
immunocyto- or immunohistochemical analysis is performed with double staining
using specific antibodies. In addition, posttranscriptional regulation may influence
specificity and transport capacity, which can be accessed by transport and inhibition
studies using cultured cell expression systems.