Flag-tagged protein showed that increasing concentra- tions of unlabeled speciﬁc competitor RNA led to the decreasing co-puriﬁcation of MADP1 with the biotinylated RNA probe (Figure 1B). However, increasing concentrations of unlabeled non-speciﬁc competitor RNA did not result in detectable change to the efﬁciency of MADP1 co-puriﬁcation (Figure 1B). Simultaneously, a protein exhibiting a non-speciﬁc RNA-binding activity, the Flag-tagged IBV-N, was used as a control. Total cell lysates containing the Flag-tagged IBV N protein was incubated with 0.1 mM of the biotinylated IBV 30-UTR, in the presence of increasing concentrations of either the unlabeled speciﬁc probe or an unlabeled non-speciﬁc probe, EGFP RNA, of an equal length. Western blot detection of the co-puriﬁed Flag-tagged N protein revealed that increasing concentra- tions of both unlabeled RNA probes increasingly reduced the efﬁciency of N protein co-puriﬁcation with the biotinylated RNA probes (Figure 1B). These results con- ﬁrmed that MADP1 could interact speciﬁcally with the 50-UTR of IBV RNA. Over-expressed Flag-tagged MADP1 translocates from the nucleus to the cytoplasm MADP1 was identiﬁed as a component of the 18 S U11/12 snRNP (37) and its subcellular localization was determined to be in the nucleoplasm (35). IBV replication Nucleic Acids Research, 2012, Vol. 40, No. 11 5069 and transcription, on the other hand, take place in the cytoplasm of the infected cells. Therefore, to validate the likelihood of MADP1 interacting with the viral 50-UTR, immunoﬂuorescence was used to track the subcellular lo- calization of both Flag-tagged MADP1 and de novo synthesized viral RNA in both mock-infected and IBV-infected cells. Flag-tagged MADP1 was over- expressed in cultured Vero cells, which were then infected with IBV and treated with actinomycin D to inhibit host transcription. The newly synthesized viral RNA, a marker for the RTCs, was labeled with BrUTP. The cells were ﬁxed at 10 h post-infection to allow sufﬁ- cient labeling of the newly synthesized viral RNA and to minimize the formation of large syncytial cells. In unin- fected cells, Flag-tagged MADP1 was localized in the nucleus exclusively (Figure 2). Upon infection by IBV, Flag-tagged MADP1 appeared to be present in the cyto- plasm as well (Figure 2). Interestingly, the cytoplasmic localization pattern of Flag-tagged MADP1 appears to be partially overlapped with that for the RTCs, although further studies would be required to ascertain if MADP1 would be a part of the RTCs (Figure 2). As a negative control for the over-expressed protein, vector transfected cells probed with Flag antibody showed negative staining for the over-expressed protein (Figure 2). Similar colocalization patterns were also observed in IBV-infected H1299 cells (Figure 2). Figure 2. Over-expressed Flag-tagged MADP1 partially colocalized with viral RTCs. Vero cells over-expressing either Flag-tagged MADP1 or empty vector were infected with IBV, treated with actinomycin D at 3 h post-infection, and transfected with bromo-UTP at 7 h post-infection. Cells were ﬁxed at 10 h post-infection and permeabilized with Triton-X 100. Immunoﬂuorescence was performed with antibodies to Flag and BrdU followed by secondary antibodies conjugated with Alexa Fluor 488 and 594, respectively. Vector-transfected Vero cells infected with IBV were used as negative controls. H122 cells transfected with Flag-tagged MADP1 and infected with IBV, as described for Vero cells.