Full-length HIP1 is overexpressed specifically in tumor tissue from TRAMP mice and is not proapoptotic. (a) Western blot analysis of normal and prostate cancer tissue from a mouse model of prostate cancer, TRAMP. Normal and prostate cancer tissue was immunoblotted using mAb’s to HIP1, clathrin heavy chain (clathrin), and adaptin-γ, a subunit of the clathrin-associated adaptor protein AP1. nl, normal prostate. (b) A mutant of HIP1 lacking a critical N-terminal ENTH domain, designated HIP1/ΔE, causes cell death. Vector (pcDNA3), full-length HIP1 (FLHIP1), HIP/ΔE (ΔE), and caspase-9 (casp-9) were transfected into 293T cells. Cell death was assayed by cellular morphology following transfection of the plasmid of interest. Western blot analysis was used to confirm expression of the various proteins. (c and d) TUNEL assays on transfected cells confirm that HIP1/ΔE–mediated cell death is apoptotic. Following transfection, cells were subjected to TUNEL assays with fluorescein-tagged dUTP and immunofluorescent labeling with HIP1 mAb. Confocal image analysis was carried out, and results for TUNEL labeling of full-length HIP1 and HIP1/ΔE –transfected cells are shown in c and d respectively, as TUNEL-positive cells (green), HIP1-immunofluorescent cells (red), and merged images. Bar graphs in c and d show the percentage of transfected cells (i.e., those staining red) that are also TUNEL-positive (green).