1. Harnessing the Ubiquitination Machinery to Target the Degradation of Specific Cellular Proteins 
Pengbo Zhou, Robert Bogacki, Lisa McReynolds, Peter M. Howley 
Molecular Cell 
Volume 6, Issue 3, Pages (September 2000)
DOI: /S (00)

2. Figure 1
Strategy for the Selective Degradation of Cellular Non-SCF Targets by Chimeric F Box Proteins
The yeast F box–containing Cdc4p normally targets the phosphorylated cyclin-dependent kinase inhibitor Sic1p for degradation. In order to target the protein of interest (Target) to the SCF machinery, the binding peptide (BP) of the intended target is covalently linked to an F box protein for directing the target to the core SCF for ubiquitination and subsequent degradation by the 26S proteasome.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Targeted Degradation of pRB by an Engineered Cdc4p-Derived Ubiquitin-Protein Ligase in Yeast
The half-life of pRB was determined by promoter shut-off in the S. cerevisiae Y81 cells (WT) under normal growth conditions (30°C) (top panel), or Y81 cells constitutively expressing Cdc4pF/WD-E7N or Cdc4pF/WD-E7N(ΔDLYC) deficient for pRB binding at 37°C (middle panels). pRB stability was also determined in the temperature-sensitive strain KY203 (cdc34-2) cells constitutively expressing Cdc4pF/WD-E7N at the nonpermissive temperature (37°C). The pRB levels at the indicated time points following promoter shut-off were determined by immunoblotting using the Ab-5 monoclonal antibody (CALBIOCHEM).
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 Degradation of pRB by the Engineered βTrCP
(A) The engineered F-TrCP-E7N ubiquitin-protein ligase can interact with pRB. In vitro translated (IVT), 35S-labeled pRB was mixed with similarly labeled IVTs οf βTrCP (lane 1), F-TrCP (lane 2), F-TrCP-E7N (lane 3), and F-TrCP-E7N (ΔDLYC) (lane 4) for 1 hr at 4°C. Complexes were then immunoprecipitated using the anti-Flag M2 monoclonal antibody and subjected to SDS–PAGE electrophoresis. Lanes 5–9 represent 50% of the input 35S-labeled proteins used in the coimmunoprecipitation analysis.
(B) The engineered F-TrCP-E7N ubiquitin-protein ligase can degrade transfected pRB. SAOS-2 cells were transiently transfected with the indicated amounts of HA-pRB and F-TrCP-E7N or F-TrCP-E7N(ΔDLYC). HA-pRB protein was determined from 200 μg of cell extracts by Western blotting using the AB-5 anti-pRB monoclonal antibody.
(C) F-TrCP-E7N blocks pRB-induced growth arrest in SAOS-2 cells. SAOS-2 cells were transfected with 1.5 μg of pBabe-puro and 5.0 μg of each of the indicated plasmids. Transfected cells were subjected to puromycin selection for 7–10 days, and the percentage of flat cells was determined. The graph shown was the average from two independent experiments.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Expression of the Engineered F-TrCP-E7N Ubiquitin-Protein Ligase Can Target Degradation of the Endogenous p107 in C33A Cells
(A) C33A cells were transiently transfected with 1 μg of pCMV-CD19 expression plasmid and the pCDNA3 vector alone, or the indicated amounts of pCDNA-F-TrCP-E7N or pCDNA-F-TrCP-E7N (ΔDLYC). Following transfection, cells were subjected to immunomagnetic selection using the anti-CD19 monoclonal antibody and Dynabeads coated with rat anti-mouse IgG1. Levels of the endogenous p107 in 200 μg of extracts of CD19-positive cells were examined by immunoblotting using the anti-p107 polyclonal antibody. The endogenous actin levels are indicated as an internal loading control.
(B) C33A cells were mock infected (C), or infected with either control adenovirus (Ad1), or adenoviruses expressing F-TrCP-E7N or F-TrCP-E7N(ΔDLYC) for 20 hr to analyze the levels of endogenous p107 as described in (A).
(C) C33A cells similarly infected by the adenoviruses as in (B) were either untreated (−) or treated (+) with 20 μM of proteasome inhibitor NLSV for 1 hr prior to the analysis of the p107 levels.
Molecular Cell 2000 6, DOI: ( /S (00) )