Identification of CARMA1 as a binding partner of CKIP-1.

(A) HEK293T cells were transfected with plasmids encoding CKIP-1 together with Myc-CARMA1 or PKCθ, lysed, and immunoprecipitated by anti-CKIP-1 or control antibody. (B) HEK293T cells were co-transfected with plasmids encoding Myc-CARMA1, PKCθ or CKIP-1, lysed, and immunoprecipitated by anti-Myc antibodies. (C) HEK293T cells were transfected with DsRed-CKIP-1. 24 hr later, the transfected cells were incubated with Alexa Flour 488-conjugated cholera toxin B (CTx), and were fixed and stained with DAPI. In lower panels, HEK293T cells were transfected with DsRed-CKIP-1 together with EGFP-CARMA1. 24 hr later, cells were fixed and stained with DAPI. The localization of CKIP-1, CARMA1 and Alexa Flour 488-CTx-labeled lipid rafts was visualized by confocal microscopy. (D) JPM50.6/WT cells, which were reconstituted by Myc-CARMA1 WT into CARMA1-deficient Jurkat T cells, were lysed and immunoprecipitated by anti-CKIP-1 or control antibody. Ig hc, immunoglobulin heavy chain. (E) Schematic diagram of CARMA1 and CKIP-1 truncated forms used in the experiments. CARD, caspase recruitment domain; CC, coiled-coil; SH3, Src homology 3; GUK, guanylate kinase; MAGUK, membrane-associated GUK; PH, pleckstrin homology; LZ, leucin zipper. (F) HEK293T cells were transfected with CKIP-1 together with Myc-CARMA1 truncated form, lysed, and immunoprecipitated by anti-Myc antibody, followed by Western blotting with indicated antibodies. (G) HEK293T cells were transfected with Myc-CARMA1 together with each CKIP-1 truncated form. Cell lysates were immunoprecipitated by anti-Myc antibody, followed by Western blotting with indicated antibodies. (H) CARMA1 CD-CC was purified from E. coli as a GST fusion protein. GST alone or GST-tagged CARMA1 CD-CC was incubated with in vitro transcribed/translated FLAG-CKIP-1. GST pull-downs and input were subjected to Western blotting with anti-FLAG antibody.