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. 2011 Jul;21(7):1152-4.
doi: 10.1038/cr.2011.89. Epub 2011 May 24.

Maximizing target protein ablation by integration of RNAi and protein knockout

Jeffrey Hannah, Pengbo Zhou

Maximizing target protein ablation by integration of RNAi and protein knockout

Jeffrey Hannah et al. Cell Res. 2011 Jul.
No abstract available

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Figures

Figure 1
Figure 1
Combining RNAi and PKO to achieve maximal and rapid protein ablation. (A) Schematic diagram of the combination approach, which entails both a reduction in nascent protein biosynthesis via RNAi as well as enhanced post-translational degradation via PKO to ablate overall steady-state protein expression levels. (B) (Top) β-TrCP-E7N chimeric E3 ligase contains the β-TrCP F-box motif to facilitate interaction with the SCF core E3 ubiquitin ligase complex and the E7N peptide, which binds Rb (as well as pocket proteins p107 and p130). (Bottom) SAOS-2 cells were transfected by nucleofection (Amaxa) with pCDNA3-Rb-HA, and either pMSCV-LMP-sh-Rb, pCDNA3-β-TrCP-E7N or both. Control cells were transfected with pMSCV-LMP. All samples were selected with puromycin 24 h post-transfection, then analyzed by immunoblotting with antibodies against Rb, FLAG or β-actin. (C) (Left) Rb-mediated inhibition of E2F1, which binds the DHFR promoter along with its cofactor DP-1, reduces transcription of the DHFR-luciferase reporter. (Right) Dual-luciferase assay (Promega) of SAOS-2 cells transfected with pCDNA3-Rb-HA, pCDNA-E2F1, pCMV-DP1, DHFR-luc, pRL-Tk (Renilla) and the indicated knockdown constructs. Cells were lysed after 24 h of puromycin selection and the ratio of Firefly/Renilla (F/R) luciferase signal was measured in triplicate. Measurements were normalized to control and the graph indicates average (of three experiments) fold difference in F/R ratio. The symbol * indicates P-value < 0.05. (D) (Top) C33A cells were transfected (Invitrogen) with anti-RBL1 siRNA oligonucleotides (Thermo Scientific), then infected with adenovirus bearing the Ad1-β-TrCP-E7N construct. °Non-specific species. (Bottom) Average p107 expression levels were obtained from three separate experiments and normalized to p107 levels in C33A cells that were mock transfected and infected.
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References

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