Nucleic Acids Research, 2012, Vol. 40, No. 11 5073 Figure 5. Knockdown of MADP1 by siRNA suppresses IBV infection. (A) RT-PCR analysis of the effect of MADP1 knockdown on IBV RNA replication. H1299 cells were transfected twice with siRNA targeting either Madp1 (+siMadp1) or EGFP (–siMadp1) and infected with IBV-Luc recombinant virus 3 days after the ﬁrst transfection. Samples were harvested at 4-h intervals, and mock-infected cells were used as negative control. RT-PCR analyses of the mRNA levels of MADP1, the negative strand IBV RNA (gRNA(-)), (+) and (–) mRNAs 3 and 4 (sgRNA) and control GAPDH were carried out. (B) Western blot analyses of the protein levels of Madp-1, viral proteins spike (S), nucleocapsid (N) and cellular protein actin for loading control. (C) TCID50 of total virus produced by the virus-infected cells showed that the silencing of Madp1 reduced virus titers by several folds. (D) Luciferase gene activity measured for the cell lysate indicated a dramatic drop in viral activity in the Madp1 silenced H1299 cells. Figure 6. Two hundred and ﬁfty pico moles (250 pmol) of either siRNA to EGFP or siRNA pools against Madp1 were transfected into H1299 cells twice and infected with recombinant luciferase-IBV at 72 h after the ﬁrst transfection. (A) Volumes (in microliters) of each 50 -mM siRNA used in the siRNA pools. (B) Luciferase activity of the infected cells measured at 20 h post-infection showed a decrease in viral activity after silencing Madp1 with the different siRNA pools.