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. 2009 Jun 24;83(17):8893–8904. doi: 10.1128/JVI.02239-08

FIG. 8.

FIG. 8.

shRNA inhibitory to CREB expression has a significant effect on viral early gene expression. HFF cells were transfected with plasmids expressing different shRNAs specific to CREB and analyzed for the steady-state level of CREB or ATF-1 by Western blotting 3 days posttransfection. GAPDH or β-tubulin served as a loading control. Viral gene expression was measured at 10 days posttransfection for MIE IE1/IE2 RNA and at 14 days posttransfection for chloramphenicol acetylation per microgram of protein relative to dlE-694/-223 DNA input as described in Materials and Methods. (A) Western blot with anti-CREB antibodies. Lanes: 1, mock-transfected cells; 2, nonspecific shRNA (N-S); 3, shRNA specific to CREB-a; 4, shRNA specific to CREB-b. (B) Western blot with anti-ATF-1 antibodies. Lanes: 1, mock-transfected cells; 2, nonspecific shRNA (N-S); 3, shRNA specific to CREB-a; 4, shRNA specific to CREB-b. (C) RT-PCR of MIE IE1/1E2 RNA at 10 days posttransfection with nonspecific shRNA (N-S) or shRNA-b specific to CREB. (D) CAT activity with nonspecific shRNA (N-S) or shRNA-b specific to CREB.