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 150

Fig. 6. Immunocytochemical staining of Flp-In-293 cells expressing ABCG2 S441N protein ( a ) and immunoelectron
microscopic image of Flp-In-293 cells expressing ABCG2 F208S protein ( b ). Cells were incubated with or without 2 m M
MG132 for 24 h. ABCG2 proteins were immunologically detected with an ABCG2-specific monoclonal antibody (BXP-21)
and Alexa Fluor 488 ( green ). Cellular nuclei were stained by Hoechst 33342 ( blue ).
non-glycosylated form (M.W. = 72,000) for both F208S and
S441N variants, where those cell lysate samples were not
treated with PNGase F (Fig. 5a ). More importantly, upon the
immunoblot analysis without PNGase F treatments, aggre-
gated forms (indicated by arrowheads in Fig. 5b ) of both
F208S and S441N variants were detected in the high molecu-
lar weight range of over 200,000.
8. It was of great interest to know how the inhibition of protea-
somal protein degradation by MG132 affects the cellular
localization of the F208S and S441N variant proteins.
Figure 6a depicts the immunofluorescence images of
Flp-In-293 cells expressing ABCG2 S441N that were incu-
bated with or without 2 mM MG132 for 24 h. In the case of
the S441N variant, MG132 treatments enhanced the local-
ization of the ABCG2 variant protein at the plasma mem-
brane and in intracellular compartments. By immunoelectron
microscopy, we could detect ABCG2 aggresome formation
adjacent to the nuclei when Flp-In-293 cells expressing the
F208 variant were treated with 2 mM MG132 for 24 h
(Fig. 6b ).
Acknowledgments
The authors thank Dr. Masako Osumi (Integrated Imaging
Research Support) and Dr. Hideaki Tamaki (Kitasato University)
for their generous support for immunoelectron microscopy
studies. Furthermore, they thank Dr. Yoichi Matsuda (Hokkaido