Journal of General Virology

Fig. 3. (A) Western blot detection of LEF-11 in infected cell extracts at various times p.i. Numbers above the lanes represent the time (h) p.i. when cell lysates were harvested. (Sf9, mock-infected cells; LEF-11, purified LEF-11 expressed from E. coli; Xf, Sf9 cells transfected with a plasmid expressing LEF-11; M, protein size standard markers). Infected cell lysates were prepared from Sf9 cells infected with AcMNPV (m.o.i.=10) and LEF-11 proteins were detected using affinity purified anti-LEF-11 antibodies (1:200 dilution) as a primary antibody and a goat anti-rabbit IgG–alkaline phosphatase conjugate as a secondary antibody (1:10000 dilution). The anti-LEF-11 antiserum was generated against His-tagged LEF-11 expressed in E. coli and purified by metal-affinity chromatography. Anti-LEF-11 antibodies were affinity purified by binding and eluting from NitroPure membranes containing the LEF-11 fusion protein, as described earlier (Monsma & Wolfner, 1988). (B) Immunofluorescent detection of LEF-11 in AcMNPV-infected Sf9 cells. Sf9 cells were infected with AcMNPV (m.o.i.=100) and fixed in methanol at 18 h p.i. Cells were then examined for LEF-11 localization using affinity purified anti-LEF-11 polyclonal antibodies (1:200 dilution) and an Alexa Fluor 594-conjugated secondary antibody. Panels (i) and (iii) show light micrographs and panels (ii) and (iv) show epifluorescence images of immunostained cells. LEF-11-specific staining (arrowheads) within dense nuclear structures in infected cells is shown. Nuclei were identified by phase contrast microscopy (i, iii). Sf9, mock-infected cells; AcMNPV 18 h p.i., infected cells at 18 h p.i.

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