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 (PP2A_C) and the structural subunit (PP2A_A). Asterisk indicates that the Flag-B overlies PP2A_A. (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-PP2A_C 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.