Expression of DNA.HTI. (A) The HTI protein is composed of 16 individual segments arranged linearly and linked via 1–3 alanine amino acid linkers and contains the GM-CSF signal peptide for better secretion. Total length of HTI protein is 529 aa, including alanine linkers. (B) Subcellular localization of HTI protein is shown in HeLa-derived HLtat cells transfected with DNA.HTI-FLAG and fixed. The HTI-FLAG protein was visualized with anti-FLAG primary antibody followed by Alexa-Fluor 488 conjugated secondary antibody and the nuclei were visualized with DAPI and a merged image is shown. (C) Plasmid DNA (250 ng) expressing HTI or p55gag with or without FLAG were transfected in HEK293 cells. The cultures were harvested after 24 hrs and 48 hrs and proteins in cell-associated (top panel: 1/100) and extra-cellular (bottom panel: 1/150) fractions were resolved on a 12% NuPAGE Bis-Tris gel. Western immunoblots were analyzed using an anti-FLAG-HRP antibody (lanes 1–4) or a goat anti-p24gag antiserum followed by anti-goat IgG-HRP labeled antibody (lanes 5–8) and were visualized using enhanced ECL. The membranes were probed with an anti-beta actin antibody to control for equal loading of the cell-associated fractions. (D) The serially diluted extracts with cell-associated and extra-cellular fractions for p55gag-FLAG and cell-associated fraction of HTI-FLAG proteins were analyzed together with FLAG-tagged p55gag protein standard. The HTI-FLAG and p55gag proteins were quantified by fitting standard curve using standard p55gag-FLAG protein. (E) Analysis of a 20-fold concentrated sample (compared to panel C) containing HTI-FLAG by Western immunoblot using anti-FLAG antibody.