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. 2009 May 7;284(27):17975–17988. doi: 10.1074/jbc.M109.012047

FIGURE 2.

FIGURE 2.

Analysis of wrky gene expression. A, RT-PCR and quantitative real time PCR analysis of wrky gene expression. RNA samples were prepared from G. lamblia wild-type non-transfected WB cells cultured in growth (Veg) or encystation medium and harvested at 24 h (Enc). RT-PCR was performed using primers specific for wrky, cwp1, and ran genes. Ribosomal RNA loading controls are shown in the bottom panel. Representative results are shown on the left. Real time PCR was preformed using primers specific for wrky, cwp1, ran, and 18 S rRNA genes. Transcript levels were normalized to 18 S rRNA levels. -Fold changes in mRNA expression are shown as the ratio of transcript levels in encysting cells relative to vegetative cells. Results are expressed as the means ± S.E. of at least three separate experiments (right). B, WRKY protein levels in different stages. The wild-type non-transfected WB cells were cultured in growth (Veg) or encystation medium for 24 h (Enc) and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-WRKY antibody. Representative results are shown. Equal amounts of proteins loaded were confirmed by SDS-PAGE and Coomassie Blue staining. C, diagrams of the pPTWRKY plasmid. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). The wrky gene is under the control of the 5′-flanking region of the α2-tubulin 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. In pPTWRKYm, a stretch of basic amino acids between residues 315 and 331 is mutated as shown in underlined letters. D and E, localization of WRKY and its mutant. The pPTWRKY or pPTWRKYm stable transfectants were cultured in growth (Veg; left panels) or encystation medium for 24 h (Enc; right panels) and then subjected to immunofluorescence analysis using anti-HA antibody for detection. The product of pPTWRKY localizes to the nuclei and cytosol in a vegetative trophozoite and to the nuclei in an encysting trophozoite, respectively (D). The product of pPTWRKYm localizes to the cytosol in both vegetative and encysting trophozoites (E).