Calmodulin directly interacts with Src in the absence and presence of Ca²⁺.

<p>(<i>A</i>) Ca²⁺-dependent calmodulin-affinity chromatography of Src solubilized from the membrane fraction of A431 cells was performed as described in Materials and Methods. The material loaded in the CaM-Sepharose column (<i>input</i>), the last fraction after washing with the Ca²⁺-buffer (<i>Ca</i>^<i>2+</i><i>-wash</i>), and the EGTA-eluted fractions were analyzed by Western blot using an anti-Src antibody. (<i>B</i>) Pull-down of Src solubilized from a membrane fraction of A431 cells was performed in the absence (-) and presence (+) of Ca²⁺ using CaM-Sepharose (<i>CaM-beads</i>) as described in Materials and Methods. Naked Sepharose 4B beads were used as negative binding control. The presence of bound Src to the beads was analyzed by Western blot using an anti-Src antibody. (<i>C</i>) Src was solubilized from a membrane fraction of A431 cells (2 mg protein) and immunoprecipitated (IP) using an anti-Src antibody as described in Materials and Methods. The immunocomplex was processed by Western blot using anti-CaM and anti-Src antibodies. Non-relevant rabbit IgG (~22 μg) was used as a negative control (mock IP).