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. 2004 Apr 22;23(10):2116–2125. doi: 10.1038/sj.emboj.7600217

Figure 4.

Figure 4

Depletion of Fbw7 by RNAi induces accumulation of c-Myc and promotes c-Myc-dependent transactivation. (A) At 48 h after transfection of HeLa cells with the indicated siRNAs, the abundance of mRNAs for Fbw7, Skp2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; internal standard) was determined by RT–PCR. (B) At 48 h after siRNA transfection, HeLa cell lysates were subjected to immunoblot analysis with antibodies to c-Myc, cyclin E, p27, Cdk2, or Hsp90 (internal standard). (C) At 48 h after siRNA transfection, HeLa cells were transfected with a vector for FLAG-c-Myc and then subjected to pulse-chase analysis. (D) At 48 h after siRNA transfection, HeLa cells were transfected with a vector for FLAG-c-Myc(T58A/S62A) and then subjected to pulse-chase analysis. (E) Activity of a c-Myc-dependent luciferase reporter gene. At 48 h after mock, Fbw7 siRNA, or Fbw7 cDNA transfection, HeLa cells were transfected with a c-Myc-dependent luciferase reporter plasmid (p4 × E-SVP-Luc). The cells were incubated for an additional 24 h and then assayed for relative luciferase activity. Data are means±s.d. of triplicates from a representative experiment. (F) Transcriptional activity of c-Myc target genes. The abundance of transcripts derived from the indicated genes (CD, carboxypeptidase D) was determined by RT and real-time PCR 72 h after siRNA transfection. Data are means±s.d. of triplicates from a representative experiment.