Figure 5

Interaction of Par-4 with NFκB and IκB in the cytoplasm of Par-4 overexpressing cells. (A) Confocal immunofluorescence of Par-4-transfected HT29 cells triple-stained for Par-4, NFκB p65 and DAPI. DAPI (blue, panel i), p65 (green, ii); Par-4, (red, iii); merge (iv) of Par-4 (red), p65 (green), Par-4/p65 colocalization (white) and DAPI; Par-4/p65 colocalization (yellow, v); overlay of Par-4/p65 colocalization (white) and DAPI (vi). Magnified images (dashed rectangles) inserted in bottom right of panels. (B) Confocal immunofluorescence on Par-4-transfected HT29 triple-stained for Par-4, IκB and DAPI (panels i-v) or triple stained for p65, IκB and DAPI (vi-x). DAPI (blue, i and vi); Par-4 (green, ii); p65 (green, iv); IκB, (red, iii and viii); merge (iv) of Par-4, IκB, Par-4/IκB colocalization (white) and DAPI; merge (ix) of p65, IκB, p65/IκB colocalization (white) and DAPI; Par-4/IκB colocalization (v); p65/IκB colocalization (x). (C) Par-4 overexpression increases Par-4 associations with NFκB and IκB, and inhibits NFκB translocation. Left panel: Confocal immunofluorescence in empty vector- and Par-4-transfected HT29 cells stained for DAPI (blue), NFκB p65 (green), Par-4 (red) and Par-4/p65 colocalization (yellow). The arrow indicates higher nuclear p65 signal in empty vector-transfected HT29 cells. Right panel: Co-immunoprecipitation of Par-4 followed by Western blot analysis of NFκB p65 and IκB in nuclear (N) and cytoplasmic (C) fractions of empty vector- and Par-4-transfected HT29 cells. The upper panels depict co-immunoprecipitation with Par-4 antibody followed by immunoblotting with p65 and IκB antibodies. Bottom panels depict protein levels of Par-4, p65 and IκB before co-immunoprecipitation.