Expression of Ac -FTT-2 and detection with anti-human 14-3-3β antibody. A. Western blot of A. caninum L3 worm lysates. Soluble protein (5 μg) from 65,000 activated and non-activated L3 lysate was separated by 4–20% gradient SDS-PAGE, followed by Western blotting with anti-human 14-3-3β antibody. Lane 1, non-activated L3 extract; lane 2, activated L3 extract. B. Expression of A. caninum V5-tagged recombinant FTT-2 in human embryonic kidney 293 cells. A cDNA encoding full length Ac-ftt-2 was cloned into vector pcDNA3.1/V5-His and transfected into HEK293 cells for expression. After 48 h, 16 μg of total protein was separated by SDS-PAGE, and proteins detected by Western blotting with anti-human 14-3-3β antibody (left panel) or anti-V5 antibody (right panel). Lanes 1 and 3, Ac-FTT-2 transfected; lanes 2 and 4, mock transfected (empty vector).C. Immunoprecipitation of A. caninum V5-tagged recombinant FTT-2 expressed in human embryonic kidney 293 cells. Lysates were prepared 48 h after transfection with pcDNA3.1/V5-His/Ac-ftt-2. Mock lysates were made from cells transfected with the empty vector alone. Recombinant Ac-FTT-2 was precipitated by the addition of anti-V5 antibody. Bound sample refers to precipitated beads, and unbound refers to supernatant removed from precipitated beads. Lane 1, Ac-ftt-2 whole lysate only (input); lane 2, Ac-ftt-2 lysate unbound sample; Lane 3, Ac-ftt-2 lysate bound sample; Lane 4, mock lysate unbound sample; Lane 5, mock lysate bound sample.