Fig 2.

siRNAs directed to enzymes involved in ganglioside biosynthesis decrease ganglioside levels, without affecting expression of other rotavirus receptors. MA104 cells were transfected with siRNAs against UGCG and GM3-s, and 72 h posttransfection parallel wells were harvested for mRNA quantification and for immunoblotting. (A) Total RNA was extracted and levels of mRNA were determined by one-step RT-PCR as described in Materials and Methods. Results are expressed as percentages relative to mRNA values obtained in cells lipofected with control siRNA (siIrr). As a negative control, the expression of endoplasmic reticulum chaperone grp94 was silenced. Means and standard deviations for results of at least three independent experiments are shown. (B) Harvested cellular proteins were resolved by SDS-PAGE, and proteins associated with rotavirus cell entry (integrin subunits α2 and β3 and hsc70 protein) were detected by immunostaining. Vimentin was used as a loading control. The result of one representative experiment of three is shown. To detect GM1a, 250 ng of total protein was applied to nitrocellulose membranes and stained using cholera toxin B subunit. Statistical analysis was done using a Student t test, and statistically significant values are shown (**, P < 0.01).