Binding of pure CaM to Rac1(K153A), Rac1(R163A), and Rac1(K153A/R163A).

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and different GST-Rac1 mutants were incubated with purified CaM (20 µg) in MOPS buffer and allowed to shake for 2 h at 4°C. GST beads were used as negative control. The incubation conditions were beads containing WT GST-Rac1 or different GST-Rac1 mutants with buffer alone, buffer plus 5 mM Ca²⁺ or buffer plus 10 mM EGTA. At the end of the incubation, beads were washed three times and bound proteins were eluted using Laemmli's sample buffer. Western blot analysis was carried out using anti-CaM antibodies. Quantification was carried out using Bio-Rad Quantity one program and *p<0.05 values were considered significantly different (n = 3). In the figure above double refers to Rac1(K153A/R163A).