Fig. 4
Complementation of the JK2 strain with the wild-type glmyb2 gene. a Diagrams of an empty vector and the complementation plasmid. (i) Empty vector contains a pac gene (puromycin resistance) under the control of a promoter of the gamma-giardin gene (Pggir) and 3′-untranslated region (3′-UTR) of the glutamate dehydrogenase gene (gdh). This plasmid has the DNA encoding the hemagglutinin (HA) tag with 3′-UTR of α-tubulin gene (α-tub). (ii) The complementation plasmid pPmyb2HA.PAC contains the glmyb2 gene with its own promoter (Pglmyb2), and GlMyb2 is expressed in an HA-tagged C-terminus form. b Western analysis. Protein extracts were prepared from trophozoites and 24 h encysting cells of various Giardia cells (WB carrying an empty vector, JK2 with an empty vector, and JK2 with the complementation plasmid) and then reacted with anti-HA antibodies. The blot was subsequently incubated with anti-GlPDI1. c Quantitative PCR (qPCR) to measure the level of glmyb2 transcript. Various Giardia cells (WB carrying an empty vector, JK2 with an empty vector, and JK2 with the complementation plasmid) incubated in encystation medium for 3, 6, and 14 h, were used to extract RNA samples. qPCR was performed using primers specific for glmyb2 genes, and transcript levels were normalized to those of the G. lamblia actin-related gene, glactin (GL50803_15113). Relative glmyb2 mRNA expression in various cells. Three independent replicates, each consisting of three technical replicates, were evaluated. Error bars represent the standard error of the mean. **P < 0.01. d IFAs showing GlCWP1 expression in various Giardia cells during encystation. Trophozoite, 14 h, 24 h encysting cells were stained with anti-GlCWP1 antibodies and then incubated with Alexa Fluor 555-conjugated anti-rat IgG. The slides were mounted with ProLong™ Gold Antifade Mountant with DAPI. They were then observed with an Axiovert 200 fluorescent microscope. Differential interference contrast (DIC) image shows cell morphology. Scale bars, 2 μm