The hemolymph from healthy Axl inhibitor shrimp and WSSV-infected shrimp were withdrawn as described previously. Hemocytes were collected by centrifugation and suspended in Leibovitz��s L-15 medium . The hemocyte suspension was then placed on cover glasses in a 4-well plate at approximately 80% confluence and incubated at 27uC for at least 30 min to allow cells to attach. The hemocytes were then washed twice with PBS, fixed with 4% paraformaldehyde in PBS at 4uC for 10 min and permeabilized with acetone at 4uC for 3 min. After blocking with 3% normal goat serum overnight at 4uC, the hemocytes were incubated at room temperature for 3 h with rabbit anti-VP15 and with mouse anti-PmFKBP46 antibody. The cells were washed with PBST three times and then reacted with FITC-conjugated goat anti-mouse IgG antibody and with Cy3 Abmole ONX-0914 dye-conjugated goat anti-rabbit IgG antibody at room temperature for 1 h. The nuclei were counterstained with TO-PRO-3 iodide and then extensively washed with PBST. The cover glasses were wet mounted and the slides were viewed using a confocal laserscanning microscope Model FV1000 . Electrophoretic mobility shift assays with protein-DNA mixtures were conducted using a protocol modified from . The reactions were performed in 15 ml of binding buffer containing approximately 300 ng of plasmid DNA and the appropriate protein to be tested. The purified proteins of PmFKBP46-His and VP15-GST were individually incubated with the plasmid DNA at 37uC for 1 h before loading onto 0.8% agarose gels. Electrophoresis was performed using TBE buffer followed by ethidium bromide staining and observation by UV transilluminator. Control proteins used in this experiment were the GST protein mentioned above and His-tagged protein purified from plasmid pET-15b containing a partial sequence of shrimp serine proteinase homologue previously reported . To test the regulative effect on DNA-binding activity of both PmFKBP46-His and VP15-GST proteins, the reactions were carried out under 2 different conditions. First, individual proteins or paired proteins were mixed with the plasmid DNA and incubated at 37uC for 1 h. Second, paired proteins were first mixed together at 37uC for 1 h before plasmid DNA was added to the reaction mixture followed by incubation for 1 h.