Fig. 3

Expression and localization of G. lamblia PLK1 (GlPLK) in G. lamblia-expressing hemagglutinin (HA)-tagged GlPLK. a A schematic diagram of plasmid pGlPLK.neo. HA-tagged GlPLK was expressed from its own promoter, Pglplk. Transfected trophozoites were selected by neomycin resistance conferred by the neo gene expressed by the Pglggi promoter, a strong promoter of the γ-giardin gene. As a control, Giardia trophozoites were also transfected with pKS-3HA.neo, a vector control. b Western blotting to examine the expression of HA-tagged GlPLK. Extracts were prepared from G. lamblia containing empty vector (lane 1) or pGlPLK.neo (lane 2) and incubated with monoclonal mouse anti-HA antibodies. Membranes were first incubated in stripping buffer and then reacted with polyclonal rat antibodies specific to protein disulfide isomerase 1 (PDI1) of G. lamblia. c Localization of GlPLK. Giardia lamblia expressing HA-tagged GlPLK was probed with mouse anti-HA antibodies. The cells were then incubated with Alexa Fluor 488-conjugated anti-mouse IgG. Slides were mounted with ProLong™ Gold Antifade Mountant with the fluorescent stain DAPI, and then examined with a Zeiss LSM700 inverted confocal laser scanning microscope. Scale bars: 2 µm. d Co-localization of GlPLK and α-tubulin in G. lamblia. Giardia cells expressing HA-tagged GlPLK were probed with rat anti-HA antibodies and mouse anti-acetylated-α-tubulin monoclonal antibodies. e Co-localization of GlPLK and G. lamblia centrin (GlCentrin) in G. lamblia. Cells were reacted with mouse anti-HA antibodies and rat anti-GlCentrin polyclonal antibodies. Cells were then incubated with Alexa Fluor 555-conjugated anti-rat IgG and Alexa Fluor 488-conjugated anti-mouse IgG. A differential interference contrast image was acquired to show cell morphology. Scale bars: 2 μm. DIC Differential interference contrast