Fig. 2

A MLF mutant with mutation residues 128–145 (enriched in basic amino acids, MLFm) showed decreased levels of the CWP1 protein and cwp1-3 and myb2 gene expression. A Diagrams of the MLF and MLFm proteins. The gray box indicates the Myelodysplasia-myeloid leukemia factor 1-interacting protein (MLF1IP) domain identified in pfam. MLFm contains mutations (underlined) of two stretches of sequences containing basic amino acids (bold) located in residues 128–145. The mlf gene was mutated and subcloned to replace the wild type mlf gene in the backbone of pPMLF (Fig. 1A), and the resulting plasmid pPMLFm was transfected into G. lamblia. B MLFm expression decreased the CWP1 level. The pPMLF and pPMLFm stable transfectants cultured in growth medium were subjected to Western blot analysis using anti-HA, anti-CWP1, and anti-Ran antibodies, respectively. The band intensity from triplicate Western blots was quantified using Image J as described in Fig. 1E. *, p < 0.05. **, p < 0.01. C Quantitative real-time PCR assays of transcript levels in the MLF and MLFm- expressing cell lines during vegetative growth. Real-time RT-PCR analysis was performed using primers specific for mlf, cwp1, cwp2, cwp3, myb2, and 18S ribosomal RNA genes, respectively. Similar levels of the 18S ribosomal RNA were detected. The mRNA levels were normalized to the 18S ribosomal RNA levels. The ratio of mRNA levels in the pPMLFm cell line to levels in the pPMLF cell line is shown and expressed as the mean ± 95% confidence intervals of at least three separate experiments. *, p < 0.05. **, p < 0.01