Analysis of IL-6 and IL-8 expression at mRNA and protein levels in IBV-infected cells stably expressing the Flag-tagged eIF-3f.

<p>a. IBV infection of Vero cell clones stably expressing of the Flag-tagged eIF3f. Two stable Vero cell clones with high level expression of the Flag-tagged eIF3f (lanes 2, 4, 6, 8 and 10) and undetectable expression of the Flag-tagged eIF3f (lanes 1, 3, 5, 7 and 9), respectively, were infected with IBV at a multiplicity of infection of 2 and harvested at 0, 8, 12, 16 and 24 hours post-infection. The expression of the Flag-tagged eIF3f was analyzed by Western blot with anti-Flag antibody. The same membrane was also probed with anti-actin antibodies as a loading control. b. Analysis of viral RNA replication and protein synthesis in IBV-infected stable cells clones with high expression of eIF3f (+eIF3f) (lanes 1–5) and undetectable expression of eIF3f (-eIF3f) (lanes 6–10). The two stable Vero cell clones were infected with IBV at a multiplicity of infection of 2 and harvested at 0, 8, 12, 16 and 24 hours post-infection. Ten micrograms of total RNA extracted from one portion of the harvested infected cells were separated on 1.3% agarose gel and transferred to a Hybond N+ membrane. Viral RNAs were probed with a Dig-labeled DNA probe corresponding to the 3′ end 680 nucleotides of the IBV genome. Bands corresponding to the genomic and subgenomic mRNA are indicated by numbers 1–6 on the right. The same membrane was also probed with a Dig-labeled GAPDH probe as loading control. The viral protein expression was analyzed by Western blot of total cell lysates prepared from another portion of the harvested infected cells with anti-IBV S protein antibodies. The same membrane was also analyzed by Western blot with anti-actin antibodies. c. Analysis of IL-8 expression. The total RNA preparations were analyzed by Northern blot with a Dig-labelled IL-8 probe (upper panels). The IL-8 expression at the protein level was analyzed by Western blot analysis with anti-IL-8 antibodies (lower panels). d. Analysis of IL-6 expression. The total RNA preparations were analyzed by Northern blot with a Dig-labelled IL-6 probe (lanes 1–8). The IL-6 expression at the protein level was analyzed by Western blot analysis of culture media (lanes 9 and 10) and total cell lysates (lanes 11–12) harvested 24 h post-infection with anti-IL-6 antibodies. Western blot analysis of the total cell lysates with anti-actin antibodies was included as a loading control (lanes 11 and 12).