FIGURE 3.

Interaction of Orai1-CMBD with CaM-N revealed by pulldown assays. A, pulldown of synthetic Orai1-CMBD by CaM-N-GST with GSH resin or by CaM-C-His₆ with nickel resin. Orai1-CMBD can be pulled down by either CaM-N or CaM-C, suggesting that in solution CaM-N also interacts with Orai1-CMBD. As a control, the peptide was added to GSH resin or nickel resin in the absence of CaM-N or CaM-C. The control experiments showed a nonspecific binding of Orai1-CMBD to GSH resin. However, this nonspecifically bound peptide was washed off the resin during the wash. B, pulldown of recombinantly expressed Orai1-CMBD or Orai1-CMBD(W76E) mutant by CaM-GST or CaM-N-GST with GSH resin. Wild type (WT) Orai1-CMBD can be pulled down by CaM or CaM-N, consistent with the pulldown results of the synthetic peptide. The W76E mutation disrupts the interaction of Orai1-CMBD with both CaM and CaM-N, suggesting that both CaM-N and CaM-C bind to Trp⁷⁶ of Orai1-CMBD.