Fig. 3

Expression of Glγ-giardin in Giardia trophozoites at G1/S- and G2-phases. a Giardia lamblia were arrested at G1/S phase with aphidicolin (Lane 1) or trophozoites at G2-phase released from aphidicolin arrest (Lane 2). Expression of Glγ-giardin was confirmed by western blot analysis. Membrane was reacted with rat anti-Glγ-giardin polyclonal antibodies (1:1000). After deprobing antibodies with stripping buffer, membrane was incubated with polyclonal rat antibodies specific to PDI1 of G. lamblia (1:10,000) as loading control. The same blot was also reacted with rat anti-Glcyclin B polyclonal antibodies (1:500). b Total RNA was prepared from G1/S-phase and G2-phase cells using TRizol. Five micrograms of RNA were converted into cDNA using an Improm-II reverse transcription system. Real-time PCR was performed using a LightCycler System and LightCycler 480 SYBR Green I Master Kit. Conditions for real-time PCR were as follows: pre-incubation at 95 °C for 5 min followed by 45 amplification cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min. Nucleotide sequences of forward and reverse primers used for real-time PCR are listed in Table 1. Normalization of mRNA quantity in the cDNA samples was performed using glactin transcript levels