Characterization of PDCD4/eIF4A Complex

Characterization of the complex PDCD4/eIF4A was evaluated by co-immunoprecipitation assay under several schemes. For these immuno-precipitation assays, cellular fractions (300 µg) were incubated with the anti-eIF4A (1:400) for 2 h at 4 °C. Next, immune complexes were precipitated with Protein G Agarose Fast Flow (Millipore) for 12 h at 4 °C, according to a previous protocol [38 (link)]. Immuno-precipitated proteins were washed 3 times and suspended in Laemmli buffer, separated by SDS-PAGE gels, and transferred to PVDF membranes for WB analysis. This same process was corroborated, but now PDCD4 was immuno-precipitated, and eIF4A detection was made.