Interaction between IKKs and the GMCSF receptor in mammalian cells. (A) Coimmunoprecipitation of endogenous proteins: HUVECs were incubated in the presence or absence of GMCSF for 10 minutes, followed by preparation of cell extracts, immunoprecipitation with anti-IKKα agarose, anti-IKKβ or control antibodies, and immunoblotting with anti-GMRα antibodies. Equal amounts of proteins were applied. (B) Colocalization of GMRα-EYFP and IKKβ-ECFP after addition of GMCSF. HeLa cells were transfected with the indicated fluorescent protein chimeras and investigated by fluorescence microscopy using filter sets discriminating between CFP and YFP fluorescence. Representative cells before (- GMCSF) and 20 minutes after addition of GMCSF (+GMCSF) are shown. (C) FRET microscopy demonstrates interaction between GMRα-EYFP and IKKβ-ECFP. HeLa cells expressing the fluorescent fusion proteins were treated with GMCSF and subjected to FRET microscopy as described in “Materials and methods.” Specific bleaching of the acceptor fluorophore (EYFP) resulted in a significant increase of the donor fluorescence (ECFP) exactly at the sites of colocalization. A ratio image of the CFP fluorescence before and after YFP photobleaching of representative cells (20 minutes after GMCSF addition) visualizes the fluorescence resonance energy transfer as indicated by arrows. Images were captured using a × 60 oil immersion objective.