Triad1 is a predominantly nuclear protein and is highly expressed in mature granulocytes and monocytes. (A) IF of Triad1-myc reveals a nuclear diffuse localization with exclusion of nucleoli in L88/5 cells.⁵³  In cells with strong nuclear staining some cytoplasmic staining is also observed. Phase-contrast image (phase contrast) indicating cellular structures are given at the right. Immunofluorescence of empty vector-transduced cells did not reveal significant staining (not shown). (B) A Triad1-specific affinity-purified antibody detects low endogenous Triad1 expression in CD34⁺ and T cells and high nuclear expression in mature granulocytes and monocytes. DNA was stained with 4′-6-diamino-2-phenylindole (DAPI) and merged pictures are indicated. Representative results from 1 of 3 unrelated healthy volunteers are indicated. Triad1 IF detects in granulocytes a ring-shaped nuclear sublocalization. Strongest Triad1 expression is observed in the DAPI dull region (arrows) within the lobes of the nuclei. As negative control, cells were stained with preimmune serum taken prior to peptide vaccination, revealing no significant signals (not shown). (C) Intracellular Triad1 was stained on fresh bone marrow (n = 1) or leukapheresis material (n = 1) and analyzed by flow cytometer yielding similar results. The figure shows the mean fluorescence of indicated compartments of the leukapheresis sample. Lowest expression was observed in CD3⁺ and CD34⁺ cells. Within the CD14⁺ fraction lowest expression was observed in the more immature fraction (Mo1) compared with mature monocytes (Mo2). Likewise, in immature myelocytic fractions (Gr1 including promyelocytes and Gr2 including myelocytes/metamyelocytes) lower expression was observed compared with mature granulocytes (Gr3). Cells were costained for Triad1 and CD45 in combination with CD34, CD3, CD14, or CD15. Within the CD14⁺ and CD15⁺ populations immature and mature compartments were defined based on CD45 staining and side scatter as described (see also Figure S2).⁵⁴  Staining with preimmune serum resulted in 30- to 100-fold lower signals (not shown).