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Fig. 2. Nuclear association of Smad1/ß-catenin/Tcf4 is increased in
TgAlk3QD kidney tissue. (A) Cellular localization in
wild-type and cystic TgAlk3QD kidney tissue.
Immunofluorescence imaging of 4 µm tissue sections was performed after
incubation with primary antibodies and fluorescein- or rhodamine-conjugated
secondary antibodies. Left panels: co-localization of Tcf4 and Smad1. In
tissue isolated from wild-type mice, Tcf4 and PSmad1 were expressed in a
cytoplasmic pattern and showed little co-localization (merge image). By
contract, in tissue isolated from TgAlk3QD mouse kidneys,
Tcf4 and P-Smad1 were expressed in a nuclear pattern with co-localization in
the majority of imaged cells (yellow). Right panels: co-localization of
ß-catenin and Tcf4. In tissue isolated from wild-type mice,
ß-catenin is expressed in a cytoplasmic pattern and demonstrates little
co-localization with Tcf4. By contrast, in tissue isolated from
TgAlk3QD mouse kidney, ß-catenin is expressed in a
nuclear pattern and co-localizes with Tcf4 in many cells (yellow). (B)
Detection of molecular complexes in cytoplasmic and nuclear fractions.
Proteins derived from cytoplasmic (actin-positive) and nuclear (acetyl
H4-positive) fractions were submitted to immunoprecipitation and
immunoblotting with specific antibodies. Quantitation of immunoblotted
proteins controlled for the quantity of immunoprecipitated protein is shown.
Molecular complexes consisting of ß-catenin and P-Smad1 were detected in
increased amounts in both cytoplasmic and nuclear fractions of
TgAlk3QD kidney tissue. Molecular complexes consisting of
ß-catenin and Tcf4 were increased in the nuclear fraction of
TgAlk3QD tissue. (C) Association of Smad1, ß-catenin
and Tcf4 with chromatin isolated from kidney tissue. Protein associations with
nuclear DNA were assessed using cisplatin crosslinking and
immunoprecipitation/immunoblotting. Molecular complexes consisting of
ß-catenin and Smad1 and of Tcf4 and Smad1 were detected in increased
quantities in TgAlk3QD versus wild-type kidney
tissues.
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