The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions
Fig 4
PET52-233 of testin directly interacts with LIM1-3 domains in vitro.
A-D) The experimental setup is similar as shown in Fig 2A. A recombinant GST-testin variant used as bait (GST-CR (A), GST-LIM1-3 (B, C) or GST-ΔPET (D)) was trapped on glutathione resin and presented in immobilised form to a second untagged testin-variant in solution used as prey (LIM1-3 (A, C), PET52-233 (B) or ΔPET (D)). Coomassie stained SDS-PAGE analysis (A-C) or western blot analysis (D) using anti-testin (green) and anti-GST (red) antibodies is shown. Input (I) shows the untagged prey protein prior to incubation with the resin. Lanes indicated with P show the proteins present on the resin (immobilized bait and/or bound prey) after the incubation. GST resin (D) or GST-cofilin resin (A-C) incubated with the same untagged testin variant as prey were used as negative controls. Untagged protein bound to the GST-testin variant immobilised on the resin is highlighted by a red box (B). Positions of bait, prey and negative control (Neg Ctr) bait are indicated in each panel, M: marker proteins (kDa). E) The indicated concentrations of GST-LIM1-3 immobilized on glutathione-sepharose beads were prepared and incubated with 2.5μM PET52-233. For each LIM1-3 concentration (indicated as [GST-LIM1-3]total (μM)), the level of unbound PET52-233 was analysed by Coomassie staining after SDS-PAGE (left). Aspecific binding was assessed by incubation of glutathione-sepharose beads, lacking LIM1-3, with a similar concentration of PET52-233 I: input, representing 2.5 μM PET52-233 in solution without incubation to LIM-1-3 coupled glutathione beads (reference for unbound 100% or bound 0%), A: aspecific binding of the PET52-233 ligand to beads without LIM1-3, M: molecular weight marker (kDa). (right) The % amounts of bound PET52-233 calculated from the measured intensities of the unbound material on gel (left) were plotted versus GST-LIM1-3 concentrations (graph right, red dots). The amount of aspecifically bound PET52-233 is represented by a black dot. The solid blue line in the graph is the fitted curve taking into account aspecific binding (see Materials and methods for details).