Fig 4. Effect of ORFV073 on NF-κB-p65 activation during ORFV infection.
(A) OFTu cells were mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073Flag (MOI = 10) and transcription of selected NF-κB regulated genes was assessed by real-time PCR at 1 and 2 h p.i. (*, P<0.05, **, P<0.01). Fold changes are relative to OV-IA82 treatment and data are mean mRNA levels from two independent experiments. (B) OFTu cells were mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073Flag (MOI = 10). Cells were fixed at 1 h p.i. and incubated with antibody against NF-κB-p65. Cells were then stained with Alexa Fluor 488-labeled secondary antibody and DAPI, and examined by confocal microscopy. Green, NF-κB-p65; blue, DAPI. (C) Cells (n = approximately 300) expressing nuclear NF-κB-p65 in each group were counted and results depicted as the mean percentage of cells containing nuclear NF-κB-p65 at the indicated time points post infection. Results are mean of two independent experiments (*, P<0.05). (D) OFTu cells were mock infected or infected with OV-IA82 or OV-IA82Δ073 (MOI = 10) and harvested at the indicated times p.i. Whole cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with antibodies against phospho or total NF-κB-p65, and GAPDH. Results in (B and D) are representative of two independent experiments.