The cFLIP_L/calmodulin interaction is mediated by the DED1 domain.

<p>Calmodulin conjugated on sepharose resin was used as bait in all pull-down assays and purified recombinant cFLIP_L or its DED domains were used as prey (10 g each). Pull-down lanes consisted of 50 μL resin and supernatant lanes of 500 μL buffer, rendering a dilution factor of 10 for non-interacting prey proteins in the SDS/Coomassie gel. <b>(A)</b> Full-length cFLIP_L pulls down on calmodulin-conjugated resin (lane 2), with DED1 strongly interacting with calmodulin (lane 3) while DED2 exhibits almost no interaction (lane 4). GST alone, used as a control, displayed no binding to calmodulin (lane 1). <b>(B)</b> The pull-down was repeated for DED1 and full-length cFLIP_L in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of Ca²⁺. Neither cFLIP nor its DED1 interact with calmodulin in the absence of Ca²⁺ (lanes 2 and 4). Only pull-down lanes are shown. <b>(C)</b> To further confirm the specificity of the DED1/calmodulin interaction, a competition assay was carried out in the pull-down format, illustrated conceptually in this schematic. Recombinant GST-DED1 is incubated with calmodulin-conjugated resin in the presence of increasing amounts of free calmodulin, which is expected to compete with the resin for binding to DED1. <b>(D)</b> Results of assay described in <b>C</b>. Free calmodulin displaces DED1 from the resin in a dose-dependent manner.