Fig. 7. B regulates the basal level of the type I receptor and B regulates its activity. (A) Outline of the experimental procedure to isolate B- and B-containing active PP2A holocomplexes, and to assay their ability to affect the kinase activity of ALK5. (B) The presence of neither PP2A complex affects the kinase activity of ALK5 in vitro. Endogenous ALK5 complexes immunopurified from untreated or TGF-β-treated HaCaT cells were incubated with recombinant Smad2 substrate in the absence or presence of B-subunit-specific PP2A complexes purified as in Fig. 6. C-terminal Smad2 phosphorylation was detected by immunoblotting. The activity of the PP2A complexes was confirmed by their ability to dephosphorylate pS259 of Raf-1 (lower panel). (C) Knockdown of B promotes ALK4 clustering. Animal caps from embryos expressing either HA-ALK4 mRNA alone (top row) or in combination with morpholino against B (MoB, middle row) were incubated for 1 hour in the presence or absence of Activin and stained with anti-HA antibody. HA-ALK4 clusters in response to Activin and in untreated embryos injected with MoB. Okadaic acid (OA) treatment (bottom row) also induces HA-ALK4 clustering and thus mimics B knockdown. (D) B knockdown strongly decreases basal protein levels of ALK5. HaCaT cells were transfected with siRNAs and treated with TGF-β as indicated. Extracts were immunoblotted with antibodies against ALK5, phospho-Smad2, pan B-subunits and Smad2/3. (E) B knockdown has no effect on TβR-II levels. HaCaT cells were transfected with the indicated siRNAs. Extracts were immunoblotted with antibodies against TβR-II, pan B-subunits and Smad2/3. Prior to electrophoresis, extracts were treated with or without PNGase F to remove N-linked sugars from TβR-II and visualise it more clearly. (F) B knockdown or B overexpression decreases protein levels of HA-ALK4. Xenopus embryos were injected at the one-cell stage with HA-ALK4 and GFP mRNAs, as well as with morpholinos or B mRNA as indicated, cultured until uninjected embryos had reached stage 9 and analysed by immunoblotting. (G) Model of the modulation of TGF-β/Activin/Nodal signalling by B and B. B normally stabilises the type I receptors ALK4 and ALK5, and B knockdown promotes their basal degradation. B normally restricts ligand-dependent activation of ALK4 and ALK5, and B knockdown facilitates such activation. When overexpressed, B additionally inhibits endogenous B by replacing it in the PP2A holoenzyme owing to its higher affinity for the catalytic subunit (not shown).