Fig. 3.
In vivo expression of GlMBP1 in G. lamblia trophozoites. (A) Quantitative measurement of GlMBP1 transcripts. Total RNA was isolated from G. lamblia using TRIzol. cDNA was synthesized from 5 µg of RNA using the ImProm-IITM RT system and then analyzed with the Light Cycler 480 II Real-Time PCR System using LightCycler 490 DNA SYBR Green I Master (Roche Applied Science). Conditions for real-time PCR were as follows: pre-incubation at 95˚C for 5 min followed by 45 amplification cycles of 95˚C for 10 sec, 56˚C for 20 sec, and 72˚C for 10 sec. Real-time PCR was carried out in triplicate in a 96-well plate using the specific primers listed in Table 1. The tim gene encoding triose-1-phosphate isomerase of G. lamblia was used as an endogenous control for the reactions. (B) Western blot analysis. Ten µg of Giardia extracts was separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with anti-GlMBP1 antibodies (1:1,000 dilution), followed by secondary antibodies (1:1,000 dilution).