H1029

RLIP76 RBD-His variants (80 nM) were immobilized on Protein A SPA fluoromicrospheres (Perkin Elmer) via an anti-His antibody (H1029, Sigma-Aldrich) in 50 mM Tris-HCl pH 7.5, 2 mM DTT, 1 mM MgCl₂, and 0.2 mg/ml BSA. [³H]GTP·Ral proteins were titrated at the concentrations indicated in the results. Experiments were performed as described previously (36 (link)). The equilibrium binding constants (K_d) for the effector–Ral interactions were determined by monitoring the SPA signal in the presence of varying concentrations of [³H]GTP·Ral and fitted using nonlinear regression with the computer program Grafit.

The Pichia pastoris KM71H-OCH protein expression system was used to produce N-terminally His and C-terminally Myc-His tagged U. maydis Erc1, Erc1^M1x, and Erc1^M2x recombinant proteins. All genes for each respective protein were cloned into the pGAPZαA vector (Invitrogen; Carlsbad, USA) under the control of a constitutive promotor with an α-factor signal peptide for secretion. Protein expression was performed by growing Pichia in 1 L buffered (100 mM sodium phosphate buffer, pH 6.0) YPD medium at 28 °C for 48 hours with 200 rpm shaking (pGAPZαA, B, & C Pichia pastoris Expression Vectors, Invitrogen; Carlsbad, USA). Recombinant protein purification was performed with a Ni-NTA-matrix (Ni-Sepharose™ 6 Fast-Flow, GE-Healthcare; Freiburg, Germany). After protein purification, each protein sample was applied to the NAP-25 column to exchange buffer with 20 mM potassium phosphate buffer pH 6.0. The proteins were stored at −20 °C for further experiments. Western blot analysis was performed by using anti-His (H1029, Sigma-Aldrich, Steinheim) and anti-Myc (M4439, Sigma-Aldrich, Steinheim) antibodies with 1:5000 and 1:3000 dilution, respectively^{20 (link)}. As a secondary antibody, anti-mouse IgG HRP (#7076, Cell Signaling Technology) was used in 1:3000 dilution^{20 (link)}.