FIG. 4.
SHARP interacts with CtIP in vivo and links CtIP with RBP-Jκ. (A) Expression of transfected SHARP-RD and CtIP was detected by Western blotting using antibodies against the FLAG (upper panel) or the Myc tags (middle panel). CtIP was coimmunoprecipitated together with SHARP-RD using an antibody directed against the FLAG-epitope exclusively from lysates where both proteins were expressed (lower panel, lane 3). Coimmunoprecipitated CtIP proteins were detected by Western blotting using an anti-Myc antibody. The asterisk indicates the heavy chain of the anti-FLAG antibody. (B, top) Either GST protein or GST-RBP-2N was immobilized on Sepharose beads and incubated with HEK-293 lysates expressing the SHARP(2002-3664) (lanes 1 and 2) or SHARP(2002-3411) (lanes 4 and 5) alone or together with Myc-CtIP protein (lanes 6, 7, 9, and 10). Only when both SHARP(2002-3664) and Myc-CtIP were expressed was a ternary complex formed with GST-RBP-2N (lane 7). This complex was not formed when a C-terminally truncated form of SHARP was expressed together with Myc-CtIP (lane 10). (B, bottom) Expression of the SHARP and CtIP proteins was verified by Western blotting. (C) HEK-293 cells were transiently transfected with an expression plasmid for SHARP(12-3664) and Myc-tagged CtIP. Cells were fixed 24 h after transfection, permeabilized, and immunostained using anti-FLAG and anti-Myc antibodies. The subcellular localization of SHARP (green, panel a) and CtIP (red, panel b) was assayed by fluorescence microscopy. (D) HEK-293 cells were transfected with various SHARP expression constructs together with Myc-CtIP as indicated. Subcellular protein localization was visualized using immunofluorescence staining, as described above. Both, wild-type SHARP (panel a) and CtIP (panel b) proteins are localized predominantly in the nucleus. Transfection of SHARP lacking the nuclear localization signal (aa 2002 to 3664, panel b) resulted in the cytoplasmic localization of CtIP (panel e). Transfection of SHARP(2002-3411) lacking both the nuclear localization signal and the RD (panel c) resulted in a restoration of the nuclear localization of CtIP (panel f).