TGFβ induces formation of endogenous complexes between Smads and PARP-1/2 in HaCaT cells.

<p>(<b>a</b>) Immunoprecipitation of Flag-Smad2/3/4 followed by immunoblotting for PARP-1 and PARP-2 in cell lysates of transiently transfected HEK-293T cells with the indicated plasmids and after stimulation with vehicle (-TGFβ) or 5 ng/ml TGFβ1 for 30 min. Expression levels of all transfected proteins and endogenous PARP-1 and PARP-2 are shown in the total cell lysate (TCL) immunoblot of the HEK 293T cells. PARP-1 immunoblot also serves as protein loading control. Stars mark non-specific protein bands. (<b>b</b>) Immunoprecipitation of Smad2/3 followed by immunoblotting for PARP-1, PARP-2, Smad2/3 and Smad4 in HaCaT cells stimulated with vehicle (-TGFβ) or with 5 ng/ml TGFβ1 for 30 min. Negative control immunoprecipitation using non-specific IgG is shown. TCL shows the levels of endogenous proteins before immunoprecipitation. PARP-1 immunoblot also serves as protein loading control and C-terminal phospho-Smad2 (p-Smad2) serves as control for the efficiency of stimulation of TGFβ signaling. (<b>c</b>) Immunoprecipitation of Smad2/3 followed by immunoblotting for PARP-1, PARP-2, Smad2/3 and Smad4 in HaCaT cells transfected with the indicated siRNAs and stimulated with 5 ng/ml TGFβ1 for 30 min or not (-TGFβ). Efficiency of knockdown of PARP-1 and PARP-2, total Smad levels, phospho-Smad2 levels and protein loading (α-tubulin) controls can be seen in the TCL. (<b>d</b>) In vitro PARylation assay after glutathion-pulldown of control GST protein or GST-Smad3, truncated mutant of GST-Smad3 (ΔMH2) and GST-Smad4 in the presence of recombinant PARP-1 and/or recombinant PARP-2 as indicated. A star (weak signal) indicates the position of PARP-2 in addition to the arrow. A longer exposure of the autoradiogram around the migrating position of PARP-2 is shown at the bottom. Note the position of ADP-ribosylated Smad proteins that migrate at the size of the core non-ADP-ribosylated proteins. The input amounts of recombinant proteins were calculated based on staining of test SDS-PAGE with CBB as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone.0103651.s001" target="_blank">Fig. S1</a>. The figure shows results from representative experiments that were repeated at least twice.