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. 2018 May 7;14(5):e1007370. doi: 10.1371/journal.pgen.1007370

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© 2018 Meyer zum Büschenfelde et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 4 — Recombinant His-tagged RIT1 wildtype (WT), p.A57G and p.F82L proteins (0.5 μM each) were loaded with GDP or non-hydrolyzable GTPγS as indicated, incubated with 1 μM GST-PAK[CRIB] bound to glutathione agarose, and precipitated. Samples were analyzed by immunoblotting using an anti-His antibody (precipitates and input) and an anti-GST antibody (precipitates). (A) RIT1 binds to PAK[CRIB]. To confirm specificity, recombinant GST-SAPAP2 (750 nM) and GST (1 μM) were used. (B) RIT1_GTPγS interacts with PAK[CRIB] in the nanomolar range. Final concentrations of His-RIT1_GTPγS are indicated in μM. As control, His-RIT1_GTPγS was incubated with GST alone (lower panels). (C) Both GDP- and GTPγS-loaded RIT1 mutants p.A57G and p.F82L interact with PAK[CRIB]. Data shown are representative of three (A and B) and two (C) independent experiments.