Effect of deletion of Rac1 putative CaM binding domain on interaction with purified bovine brain CaM.

<p>Equal amount (20 µg) of wild type (WT) GST-Rac1 and GST-Rac1 mutant (amino acids 151 to 164 deleted) 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 WT GST-Rac1 or GST-Rac1 mutant beads 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. A representative autoradiograph and quantitation is shown above. The experiment was repeated a minimum of three times.