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Fig. 6. B
and B
do not act directly on
phosphorylated Smad2. (A) Outline of the experimental procedure to
isolate B- and B-containing active PP2A holocomplexes and to
perform phosphatase assays. (B) Silver-stained gel showing the
composition of complexes isolated by Flag pulldown from HeLa cells transfected
with the indicated Flag-tagged B subunits. The components of the complex are
indicated including the catalytic subunit (PP2AC) and the
structural subunit (PP2AA). Asterisk indicates that the
Flag-B overlies PP2AA. (C) Western blot analysis of
immunopurified complexes showing the presence of appropriate B or B'
(PPP2R5D) subunit (Flag blot) and co-purified catalytic subunit
(anti-PP2AC blot) for each complex. Phosphatase activity was
assessed by a colorimetric assay using a phospho-peptide as substrate (bars).
(D) PP2A complexes (as in C) were incubated with phospho-Smad2
immunopurified from TGF-β-induced HaCaT EGFP-Smad2 cells. The reactions
were then analysed by immunoblotting with anti-phospho-Smad2 and anti-Smad2/3
antibodies. All PP2A complexes tested failed to dephosphorylate phospho-Smad2.
B- and B-containing complexes dephosphorylated pS259 of
immunoprecipitated HA-tagged Raf-1 (lower panels). (E) TGF-β
treatment prior to immunopurification of the PP2A complexes does not affect
the amount of co-purified catalytic subunit, nor the activity of the complexes
in the colorimetric assay. (F) As in D, but PP2A complexes were
purified from untreated (-) or TGF-β-induced (+) cells, as shown in E.
(G) Phosphorylated serines 245, 250 and 255 of Smad2 are not substrates
for immunopurified B and B complexes. Phosphatase complexes were
immunopurified from either control cells (C) or cells expressing Flag-tagged
B or B as indicated, and incubated with either a Smad2/3
immunoprecipitate from TGF-β-induced HaCaT cells (upper panels) or, as a
control, an immunopurified phosphorylated Raf substrate from HeLa cells
expressing HA-Raf (lower panel). Samples were analysed by western blotting
using antibodies recognising Smad2 phosphorylated at residues S245, S250,
S255, as well as anti-Smad2/3, anti-phospho Raf and anti-HA as indicated.
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