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Figure 6. The C619Y Mutation Alters the Hormone-Dependent
Subnuclear Localization of the AR
Immunofluorescence was performed on HeLa cells transfected with WT or
C619Y receptors. Panels A (WT) and D (619Y) show untreated cells
immunostained with anti-AR and a FITC-conjugated secondary antibody.
Both WT and C619Y have a diffuse and predominantly cytoplasmic
distribution in the absence of hormone. Panels B (WT) and E (C619Y)
show cells treated 16 h with 5 nM R1881. WT receptor
translocates to the nucleus and adopts a hyperspeckled distribution in
response to hormone. C619Y also translocates to the nucleus in response
to hormone but forms intranuclear aggregates of varying size that are
substantially different from foci containing WT receptor. Nuclear
aggregates containing C619Y range in size and can be up to 2 µm in
diameter. C619Y aggregates are found in almost all cells with
detectable levels of immunofluorescence, suggesting that aggregates do
not result from overexpression. In some cells cytoplasmic C619Y
aggregates can be seen. To demonstrate that C619Y aggregates are not a
staining or fixation artifact, GFP fusions of WT and C619Y were
transfected into HeLa cells and microscopy was performed on live,
unfixed cells. Panels C (GFP-WT) and F (GFP-C619Y) show live cells
after hormone treatment. Both GFP-WT and GFP-C619Y localize similarly
to their immunostained, untagged counterparts. Bar
= 10 µm.
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was not a
faithful reproduction of the original figure due to printer error. The
original high-quality figure and legend are reprinted below. The
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