Figure 2. Binding affinity and specificity of Phox-I1 to p67^phox.

(A) Using microscale thermophoresis, p67^phox recombinant protein (1–200) was able to bind Phox-I1 with an Kd of ~100nM. (B) Similar to A, the ability of Phox-I1 to bind a recombinant mutant of p67^phox at the site critical for Rac1-GTP binding, p67R38Q, was tested by microscale thermophoresis. (C) Experiment described in B was repeated with a random p67^phox mutation, R188A. (D) Constitutively active Rac1V12 mutant protein binds p67^phox with an Kd of ~40 nM using microscale thermophoresis. (E) However, Rac1 wild type protein (predominantly in GDP- bound state) cannot bind p67^phox using microscale thermophoresis. (F) Competitive binding of p67^phox with Phox-I1 or vehicle control, followed by titration of RacV12 protein using above methods. (G) Phox-I1 is unable to bind RacV12 recombinant protein via above technique.