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. 2005 Feb;25(4):1283–1297. doi: 10.1128/MCB.25.4.1283-1297.2005

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Copyright © 2005, American Society for Microbiology

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FIG. 5. — PTB1 and PTB4 enhance the IRES activity of p27 mRNA in vivo. (A) Schematic diagram of dicistronic mRNAs used for in vivo monitoring of the effect of PTB on IRES activities. The dicistronic mRNA containing the HCV IRES was used as a control. (B, i) 293T cells were cotransfected with a reporter plasmid (pR/p27/F or pR/HCV/F), an effector plasmid expressing GFP, GFP-PTB1, or GFP-PTB4, and the control plasmid pCMV•SPORT-βgal. Forty-eight hours posttransfection, cells were harvested and luciferase activities were measured. Firefly and Renilla luciferase activities were normalized with β-galactosidase activity to adjust transfection efficiency. Black and white bars depict firefly and Renilla luciferase activities, respectively. Luciferase activities in cells expressing GFP is set to 1 (lanes 1 and 4). (ii) Lysates were Western blotted with a monoclonal anti-GFP antibody. (iii) Northern blot analysis of reporter dicistronic mRNA R/p27/F produced in the transfected cells. Three micrograms of poly(A)⁺ RNAs purified from transfected cells were subjected to Northern blotting with a ³²P-labeled probe corresponding to the firefly luciferase gene. The positions of 28S and 18S rRNAs are indicated by 28S and 18S, respectively. The hRPL32 blot was used as an internal control of poly(A)⁺ mRNAs. The arrow indicates the position of the reporter mRNA. Radioactivities of the bands corresponding to dicistronic reporter mRNA (R/p27/F) were normalized to those corresponding to hRPL32 mRNAs. Numbers under the panel reporter/hRPL32 indicate the relative radioactivities of dicistronic reporter mRNA (R/p27/F) bands normalized with hPRL32 bands. The value of mRNAs from the cells expressing GFP was set to 1 (lane 2). (C) 293T cells were cotransfected with reporter plasmid pR/p27/F and effector plasmids expressing GFP (lane GFP) and ITAFs fused with GFP. The identities of the various ITAFs are indicated below the graph (i) and the gel (ii). Forty-eight hours posttransfection, the relative luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity in cells transfected with effector GFP was set to 1 (i). (ii) Lysates were Western blotted with a polyclonal anti-GFP antibody.

FIG. 5. — PTB1 and PTB4 enhance the IRES activity of p27 mRNA in vivo. (A) Schematic diagram of dicistronic mRNAs used for in vivo monitoring of the effect of PTB on IRES activities. The dicistronic mRNA containing the HCV IRES was used as a control. (B, i) 293T cells were cotransfected with a reporter plasmid (pR/p27/F or pR/HCV/F), an effector plasmid expressing GFP, GFP-PTB1, or GFP-PTB4, and the control plasmid pCMV•SPORT-βgal. Forty-eight hours posttransfection, cells were harvested and luciferase activities were measured. Firefly and Renilla luciferase activities were normalized with β-galactosidase activity to adjust transfection efficiency. Black and white bars depict firefly and Renilla luciferase activities, respectively. Luciferase activities in cells expressing GFP is set to 1 (lanes 1 and 4). (ii) Lysates were Western blotted with a monoclonal anti-GFP antibody. (iii) Northern blot analysis of reporter dicistronic mRNA R/p27/F produced in the transfected cells. Three micrograms of poly(A)⁺ RNAs purified from transfected cells were subjected to Northern blotting with a ³²P-labeled probe corresponding to the firefly luciferase gene. The positions of 28S and 18S rRNAs are indicated by 28S and 18S, respectively. The hRPL32 blot was used as an internal control of poly(A)⁺ mRNAs. The arrow indicates the position of the reporter mRNA. Radioactivities of the bands corresponding to dicistronic reporter mRNA (R/p27/F) were normalized to those corresponding to hRPL32 mRNAs. Numbers under the panel reporter/hRPL32 indicate the relative radioactivities of dicistronic reporter mRNA (R/p27/F) bands normalized with hPRL32 bands. The value of mRNAs from the cells expressing GFP was set to 1 (lane 2). (C) 293T cells were cotransfected with reporter plasmid pR/p27/F and effector plasmids expressing GFP (lane GFP) and ITAFs fused with GFP. The identities of the various ITAFs are indicated below the graph (i) and the gel (ii). Forty-eight hours posttransfection, the relative luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity in cells transfected with effector GFP was set to 1 (i). (ii) Lysates were Western blotted with a polyclonal anti-GFP antibody.