Fig. 1

Colocalization and interaction of Giardia MLF and hMLF2. A Diagrams of the 5′5N-Pac, pPMLF and pPhMLF2 plasmids. The pac gene (open box) is under the control of the 5′- and 3′ -flanking regions of the gdh gene (striated box). In constructs pPMLF and pPhMLF2, the mlf and hmlf2 genes are under the control of the 5′ -flanking region of the mlf gene (open box) and the 3′ -flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. B Immunofluorescence analysis of hMLF2 distribution. The pPhMLF2 stable transfectants were cultured in growth (Veg, vegetative growth, upper panels) or encystation medium for 24 h (Enc, encystation, lower panels) and then subjected to immunofluorescence analysis using anti-HA antibody for detection. C Colocalization of MLF and hMLF2. The pPhMLF2 stable transfectants were cultured in growth (Veg, vegetative growth) and then subjected to immunofluorescence assay. The endogenous Giardia MLF protein and vector expressed HA-tagged hMLF2 protein were detected by anti-MLF and anti-HA antibodies, respectively. D Quantification of hMLF2 vesicles in pPhMLF2 cell line during vegetative and encysting stages using Imaris software. *, p < 0.05 (n = 200–300 cells/condition). E Expression of hMLF2 increased the CWP1 protein level. The 5’Δ5N-Pac, pPMLF, and pPhMLF2 stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-HA, anti-CWP1, anti-MLF, and anti-RAN antibodies, respectively. Equal amounts of protein loading were confirmed by SDS-PAGE with Coomassie Blue staining. A similar level of the RAN protein was detected. The intensity of bands from three Western blot assays was quantified using Image J. The ratio of each target protein over the loading control RAN is calculated. Fold change is calculated as the ratio of the difference between pPMLF/pPhMLF2 cell line and the control cell line, to which a value of 1 was assigned. Results are expressed as mean ± 95% confidence intervals. *, p < 0.05. **, p < 0.01. F hMLF2 expression increased cyst formation. The 5’Δ5N-Pac, pPMLF, and pPhMLF2 stable transfectants were cultured in growth medium and then subjected to cyst count. The sum of total cysts is expressed as relative expression level over control. Values are shown as means ± 95% confidence intervals. *, p < 0.05. G Interaction between Giardia MLF and hMLF2. Expression of HA-tagged hMLF2 and MLF proteins was detected in whole cell extracts for co-immunoprecipitation assays (Input, upper panel). The 5’Δ5N-Pac and pPhMLF2 stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot. The blot was probed with anti-HA, anti-MLF, and anti- RAN antibodies, respectively. Interaction between hMLF2 and MLF was detected by co-immunoprecipitation assays (bottom panel). The 5’Δ5N-Pac and pPhMLF2 stable transfectants were cultured in growth medium. Proteins from cell lysates were immunoprecipitated using anti-HA antibody conjugated to beads. The precipitates were analyzed by Western blotting with anti-HA, anti-MLF, and anti-RAN antibodies, respectively, as indicated. H Confirmation of interaction between hMLF2 and MLF. The pPhMLF2 stable transfectants were cultured in growth medium. Proteins from cell lysates were immunoprecipitated using anti-MLF antibody to assess binding of MLF to hMLF2. Preimmune serum was used as a negative control. The precipitates were analyzed by Western blotting with anti-MLF, and anti-HA antibodies, respectively, as indicated. I hMLF2 protein was not recognize by anti-MLF antibody. Purified V5-tagged hMLF2 protein was analyzed by Western blotting with anti-V5 and anti-MLF, respectively, as indicated