APH-2 localizes to the nucleus. (A) Amino acid sequence of the APH-2 protein. The amino acid sequence of APH-2 was deduced from the MO strain. Underlining represents the peptides used for the immunization of the rabbits; dashed boxes, 2 potential LXXLL motifs. (B) Cell lysates (50 μg) from 293T cells transfected with the APH-2-Myc or HBZ-SP1-Myc expression vector or 2 control empty vectors (parental pcDNA3.1/Myc and pSG5M vectors) were subjected to electrophoresis on a 12% Bis-Tris gel and analyzed by Western blot with anti-Myc, anti–APH-2, or anti–β-tubulin antibodies. pcDNA3.1/Myc parental vector was used as a negative control. (C-E) Intracellular localization of APH-2 in (C-D) HeLa and (E) Jurkat or primary T lymphocytes transfected with HBZ or APH-2 expression vectors. (Ci-iv) APH-2-Myc. (Cv-viii) HBZ-SP1-Myc. (D-E) GFP-APH-2. Twenty-four hours after transfection, cells were fixed and stained as described in “Immunofluorescence analyses.” Cells were finally mounted in 4,6-diamidino-2-phenylindole-containing mounting medium. Images are representative of the entire population of transfected cells.