S100A4, as a substrate of TG2 also binds directly with TG2.

<p>(<b>A</b>) <b>Co-immunoprecipitation assay to detect the interaction between S100A4 and TG2.</b> After incubating recombinant S100A4 and gplTG at 37°C for 1 h, co-IP was performed. Briefly, specific antibodies, against S100A4 and TG2, respectively, were bound to the resin beads before incubation with the protein mixture to form the antibody-antigen (bait protein, S100A4 or TG2) complex. Mouse IgG and rabbit IgG were used as the negative control antibody (negative CNTL ab) for anti-TG2 antibody Cub7402 and rabbit anti-S100A4 antibody, respectively. Western blotting was carried out to detect the presence of prey proteins (TG2 and S100A4 in this case) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057017#s2" target="_blank">Materials and Methods</a>. (<b>B</b>) <b>The interaction between S100A4 and TG2 via Far Western blotting.</b> PVDF membranes bound with rhS100A4 or gplTG were prepared via Western blotting after SDS-PAGE. The membranes containing the prey protein (either S100A4 or TG2) were incubated with their relevant bait protein (gplTG or rhS100A4, respectively) as introduced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057017#s2" target="_blank">Materials and Methods</a> and Western blotting was performed to detect the presence of the bait proteins by using their specific antibodies, i.e. Cub7402 and rabbit anti-S100A4 antibody, respectively. (<b>C</b>) <b><i>In vitro</i></b><b> cross-linking assay to show S100A4 is a TG2 substrate.</b> Recombinant S100A4 (after cleavage of the his-tags) and gplTG at the concentrations in µg shown were incubated in the presence of Ca²⁺ and DTT at 37°C. The TG2 inhibitor or the Ca²⁺ chelator EDTA were used as the negative controls. The presence of S100A4 polymers was detected by using Western blotting as introduced in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057017#s2" target="_blank">Materials and Methods</a>.