Fig.6. CHIP assay
Vero cells, in triplicates, were infected with BPXV at an MOI of 5, followed by washing with PBS and addition of serum-free DMEM supplemented with hesperetin or vehicle control. At 16 hpi, cell lysates were prepared as described in method section and incubated with α-eIF4E (reactive antibody), α- MNK1 (nonreactive antibody) or equivalent volume of IP buffer (Beads control). This was followed by incubation with a Protein A Sepharose® slurry. The beads were washed with IP buffer, and cross-linking was reversed using Proteinase K. The reaction mixture was centrifuged and the resulting supernatant was subjected to RNA isolation, cDNA preparation, and quantitation of BPXV M gene by qRT-PCR. Values are representative of three independent separate experiments. Error bars indicate SD. Pair-wise statistical comparisons were performed using the Student’s t test (*** = P<0.001).