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. 2010 Oct 20;85(1):568–581. doi: 10.1128/JVI.01611-10

FIG. 2.

FIG. 2.

The 125-kDa ORF66-specific PKA substrate protein is a host cell protein. MRC-5 cells were coinfected with replication-deficient adenoviruses: Ad.Toff, which expresses a tetracycline-controlled transactivator, at an MOI of 2.5, and either mock infection or Ad.GFP-66 (expressing functional ORF66) (Ad.66), Ad.GFP-66kd (expressing kinase-inactive ORF66) (Ad.66kd), or Ad.Vector, at an MOI of 5. Doxycycline was added (+) to turn off ORF66/66kd gene expression. Cells were harvested 1 day postinfection, and immunoblot analysis was performed on cell extracts with a rabbit α-p-PKA substrate antibody. The blot was stripped and reprobed with mouse α-HA to analyze for ORF66/66kd expression. Mouse α-tubulin was used to evaluate equal amounts of total protein levels. The star marks a nonspecific protein species that was present under all conditions. The arrow indicates the ORF66-dependent 125-kDA p-PKA substrate band. Protein size markers (in kilodaltons) are specified to the left.