FIG. 5.

Overexpression of HA-Myb1 in T. vaginalis. An HA-Myb1 protein expression plasmid, pFLPha-myb1/TUBneo (A), was used to overexpress HA-Myb1 in T. vaginalis T1 cells. In this plasmid, the flp-1 promoter (FLP) drives an HA-tagged myb1 gene, and the β-tubulin (TUB) promoter drives a selective marker, the neo gene. (B) Subcellular localization of HA-Myb1 in pFLPha-myb1/TUBneo-transfected cells was detected using a mouse monoclonal anti-HA antibody and a FITC-conjugated secondary antibody. Images of cells labeled DAPI (4′,6′-diamidino-2-phenylindole), FITC, and phase contrast were recorded by confocal microscopy. (C) Lysates from nontransfected cells (lanes 1, 3, and 5) or cells transfected with pFLPha-myb1/TUBneo (lanes 2, 4, and 6) in normal medium for 8 h were examined by Western blotting using rat monoclonal anti-HA (lanes 1 and 2), anti-Myb1 (lanes 3 and 4), anti-malic enzyme (lanes 5 and 6, upper panels), or anti-α-tubulin (lanes 5 and 6, lower panels) antibody.