Fig 2: The RAM domain of NOTCH1 mediates its interaction with CARMA1. (A) GFP–N1IC mutant expression plasmids were transfected into 293T cells and sub-cellular localization of NOTCH1 was examined using fluorescence microscopy. (B) VSV–CARMA1 expression plasmids were transiently co-transfected with GFP empty vector as a control, or with GFP–N1IC mutant plasmids in 293T cells, as indicated. Transfected cells were harvested and cell lysates were co-immunoprecipitated with antibodies specific for GFP. Co-immunoprecipitated proteins were then immunoblotted with anti-VSV or anti-GFP. Ig heavy chain indicates immunoglobulin heavy chain.

Fig 3: NOTCH1 interacts with CARMA1 and BCL10 following stimulation with anti-CD3e and anti-CD28. (A) Jurkat T cells were stimulated with plate-bound anti-human CD3e and anti-human CD28 and subjected to sucrose gradient centrifugation. All fractions were analyzed by dot blot with anti-cholera toxin-conjugated HRP (upper panel). Pooled fractions were immunoblotted with indicated antibodies (lower panel). (B) Jurkat T cells were stimulated as in (A) for the indicated time periods, followed by co-immunoprecipitation with antibodies specific for NOTCH1, CARMA1, or BCL10. Eluates were subjected to immunoblotting with indicated antibodies. 1/100 of input (shown) was immunoblotted with anti-CARMA1, anti-BCL10, and anti-NOTCH1. Arrow heads beside IB: NOTCH1 represent 120 kDa, transmembrane form (upper arrow head) and 110 kDa intracellular form (lower arrow head) of NOTCH1. Data are representative of at least three independent experiments.

Fig 4: Cytosolic N1IC can interact with CARMA1 in a bifluorescence complementation assay. Jurkat T cells were transfected with various constructs of nuclear and/or cytosolic proteins and their physical association was assessed microscopically by their ability to reconstitute two halves of a yellow fluorescent protein reporter. Twenty-four hours after transfection, cells were plated onto glass bottom culture dishes, stimulated with anti-human CD3e and anti-human CD28, and fluorescent images of live cells were captured using a Zeiss LSM 510 confocal microscope. (A) Nuclear proteins, cFos and cJun, known to interact in the nucleus were co-expressed as a control; (B) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was co-expressed with nuclear cJun; (C) NOTCH1 with an additional nuclear export signal, N1IC–NES, was co-expressed with nuclear cJun; (D) NOTCH1 with an additional nuclear export signal, N1IC–NES was co-expressed with CARMA1, a cytosolic protein; (E) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was CARMA1; (F) NOTCH1 with an additional nuclear export signal, N1IC–NES was expressed alone. Upper panel of all images represent fluorescent channel only; lower panel of all images represent merged fluorescent and DIC images. Data are representative of at least three separate experiments.