1. Inhibitor Mediated Protein Degradation
Marcus J.C. Long, Deviprasad R. Gollapalli, Lizbeth Hedstrom 
Chemistry & Biology 
Volume 19, Issue 5, Pages (May 2012)
DOI: /j.chembiol
Copyright © 2012 Elsevier Ltd Terms and Conditions

2. Chemistry & Biology 2012 19, 629-637DOI: (10. 1016/j. chembiol. 2012
Copyright © 2012 Elsevier Ltd Terms and Conditions

3. Figure 1 Degradation of HA-Tagged GST-α1
(A) Structures of EA and EA-Boc3Arg and the reaction of GST with EA derivatives.
(B) EA and EA-Boc3Arg inactivate GST-α1 with similar potency. Purified recombinant GST-α1 (200 nM) was incubated with EA or EA-Boc3Arg and assayed for activity.
(C and D) The degradation of EA-Boc3Arg-modified GST-α1. Purified recombinant C-terminally HA-tagged GST-α1 (0.2 mg/ml) was inactivated by EA or EA-Boc3Arg (80 μM) and diluted 50-fold into HeLa cell lysates supplemented with an ATP regenerating system. Samples were analyzed by immunoblotting for the HA tag. Inosine monophosphate dehydrogenase (IMPDH) was used as a loading control. (C) A representative immunoblot. (D) Average of four experiments. Error bars show standard errors.
(E) Structure of Fur-Boc3Arg.
(F) Degradation of GST-α1 by Fur-Boc3Arg. Purified C-terminally HA-tagged GST-α1 was modified with Fur-Boc3Arg (40 μM) and added to NIH 3T3 cell lysates supplemented with an ATP regenerating system.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2012 Elsevier Ltd Terms and Conditions

4. Figure 2
TMP-Boc3Arg Induced Degradation of HA-Tagged eDHFR in Cell Lysates
(A) Structures of TMP and derivatives.
(B and C) Purified recombinant eDHFR-HA (0.2 mg/ml) was incubated with TMP derivatives (80 μM) and diluted 50-fold into Cos-1 cell lysates supplemented with an ATP regenerating system. (B) Representative immunoblot showing degradation of eDHFR-HA in the presence of TMP and TMP-Boc3Arg. (C) Combined data from two experiments after 4 hr incubation. Bar graphs show the average and the range of values.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2012 Elsevier Ltd Terms and Conditions

5. Figure 3 Degradation of eDHFR Fusion Proteins in Whole Cells
(A and B) Global protein stability assay. HeLa cells co-express RFP and eDHFR-HA-GFP from a bicistronic construct. Red and green fluorescence was measured by flow cytometry. (A) TMP (80 μM). (B) TMP-Boc3Arg (80 μM).
(C) Quantitation of four independent GPS experiments. Bar graphs show the average and the standard deviation.
(D) Degradation of eDHFR-GFP was confirmed by anti-HA and anti-GFP immunoblotting.
See also Figure S1.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2012 Elsevier Ltd Terms and Conditions

6. Figure 4
Degradation of eDHFR Fusion Proteins in Cycloheximide-Treated Cells
(A) Experiment as in Figure 3, but HeLa cells were incubated with 0.2 mg/ml cycloheximide for 20 min prior to TMP-Boc3Arg treatment. The rightmost panel is an independent blot.
(B) Quantitation of three independent experiments. Bar graphs show the average and the standard deviation.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2012 Elsevier Ltd Terms and Conditions

7. Figure 5
EA-Boc3Arg-Induced Degradation of Endogenous and Ectopic GST in Whole Cells
(A) Degradation of endogenous GST-π when Cos-1 cells are treated with EA and EA-Boc3Arg for 3 hr (n = 4). Bar graphs show the average and the standard deviation.
(B) Degradation of eDHFR-HA-GST-α1 in HeLa cells. Cells expressing eDHFR-HA-GST-α1 were treated with EA-Boc3Arg (80 μM) over 1.8 hr. Protein was measured by both anti-GST and anti-HA immunoblotting (n ≥ 3). Bar graphs show the average and the standard deviation.
(C) HeLa cells expressing eDHFR-HA-GST-α1 were treated with: triangles, EA (80 μM); squares, EA-Boc3Arg (8 μM); and diamonds, EA-Boc3Arg (80 μM). At the indicated time points, the eDHFR-HA-GST-α1 was quantitated and normalized to GAPDH (n = 2). Bar graphs show the average and the range of values.
(D) HeLa cells expressing eDHFR-HA-GST-α1 were treated with a range of concentrations of EA-Boc3Arg or just dimethyl sulfoxide. After 1.2 hr, the amount of eDHFR-HA-GST-α1 was quantitated (n = 2). Bar graphs show the average and the range of values.
See also Figure S2.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2012 Elsevier Ltd Terms and Conditions

8. Figure 6 Mechanism of Boc3Arg-Induced Degradation
(A) Effect of lactacystin (50 μM) on the TMP-Boc3Arg-induced degradation of eDHFR in Cos-1 cell lysates supplemented with an ATP regeneration system.
(B) Degradation of GST-α1 (0.2 mg/ml) was modified by EA-Fur-Boc3Arg (40 μM) then diluted 50-fold into NIH 3T3 cell lysates. Closed diamonds, standard assay buffer containing ATP regenerating system; closed squares, ATP regenerating system omitted.
(C) Effect of lactacystin (50 μM) and ubiquitin aldehyde (20 μM) on the degradation of eDHFR-TMP-Boc3Arg in Cos-1 lysates.
(D) Effect of MG132 (100 μM) on the EA-Boc3Arg-induced degradation of GST-α1 in Cos-1 cell lysates supplemented with an ATP regeneration system.
Chemistry & Biology  , DOI: ( /j.chembiol )
Copyright © 2012 Elsevier Ltd Terms and Conditions