1. Volume 18, Issue 5, Pages 565-576 (May 2005)
Loss of HAUSP-Mediated Deubiquitination Contributes to DNA Damage-Induced Destabilization of Hdmx and Hdm2 
Erik Meulmeester, Madelon M. Maurice, Chris Boutell, Amina F.A.S. Teunisse, Huib Ovaa, Tsion E. Abraham, Roeland W. Dirks, Aart G. Jochemsen 
Molecular Cell 
Volume 18, Issue 5, Pages (May 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 HAUSP Stabilizes p53, Hdm2, and Hdmx
(A) U2OS cells (6-well plates) were transfected with expression vectors for HA-p53 (200 ng), with or without Hdm2 (500 ng), in absence or presence of 2 μg HAUSP. Western blot was performed on total lysates of the cells harvested 40 hr after transfection. Hdm2 and p53 were detected with the anti-HA antibody; HAUSP was detected with the mouse monoclonal antibody 1G7. Tubulin expression was analyzed as loading control.
(B and C) U2OS, H1299 cells were transfected as indicated by using 500 ng HA-Hdmx, 200 ng HA-Hdm2, 1 μg or 3 μg HAUSP, and 3 μg HAUSP (C223S) expression vectors. Hdm2 and HAUSP were detected as in (A); Hdmx was detected with the anti-HA antibody.
(D) LS89 cells were incubated with doxycycline (1 μg/ml) for the indicated time points. HAUSP was detected with the monoclonal anti-HAUSP antibody 1G7, Hdm2 with 4B2, Hdmx with 6B1A, and p53 with DO-1. Tubulin was analyzed as a loading control.
(E) LS89 cells were treated with doxycycline as in (D). RT-PCR was performed on RNA isolated at the indicated time points.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
HAUSP Expression Is Essential for Maintenance of Hdmx Protein Level
(A) U2OS cells (6 cm dishes) were transfected with 5 μg of the indicated pSUPER plasmids. Cells were harvested 72 hr after transfection. The expression of HAUSP, Hdm2, Hdmx, and p53 and tubulin as a loading control were analyzed by Western blot as in Figure 1D.
(B) HAUSP RNAi-inducible cells (LS88) were incubated with doxycycline (1 μg/ml) for the indicated time points. Total cell extracts were analyzed for the expression of the indicated proteins as described in Figure 1D.
(C) HAUSP RNAi-inducible cells (LS88) were either incubated with doxycycline (1 μg/ml) for 48 hr or mock treated; subsequently, the cells were incubated for the indicated time points with cycloheximide (50 μg/μl).
(D) Total cell extracts of control HCT116 cells and HCT116 HAUSP−/− cells were analyzed as in Figure 1D.
(E) U2OS cells were transfected with HA-Hdmx (200 ng) or HA-p53 (50 ng) expression vectors, without or with increasing amounts of HA-Hdm2 (100 ng, 200 ng, and 400 ng) expression plasmid. The expression of HA-Hdmx, HA-Hdm2, and HA-p53 proteins was analyzed by Western blotting with the use of anti-HA.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3 HAUSP Interacts with Hdmx
(A) U2OS cells (9 cm dishes) were transfected with 5 μg HA-Hdmx and/or 5 μg Myc-HAUSP. The Hdmx proteins were immunoprecipitated with a combination of the anti-Hdmx antibodies p55/p56, and the immunoprecipitates were analyzed by Western blot, with use of the anti-Myc antibody to detect HAUSP. Myc-tagged HAUSP was immunoprecipitated with anti-Myc antibody, and HA-Hdmx proteins were detected with the anti-HA antibody.
(B) GST-Hdmx, GST-Mdm2, or GST only were incubated with 35S-methionine-labeled, in vitro-translated HAUSP; 10% of the input was loaded as a control.
(C) Schematic representations of different splicing variants of Hdmx.
(D) Transfection of U2OS cells with 5 μg Myc-HAUSP and/or 5 μg of the HA-Hdmx splicing variant expression vectors. Immunoprecipitations were performed with the anti-Myc antibody, after which the immunoprecipitates were analyzed with anti-HA. To be able to detect full length Hdmx and Hdmx-E simultaneously, samples were separated on a 15% SDS polyacrylamide gel.
(E) Cell extracts from C33A, MCF-7, and U2OS cells were analyzed by immunoprecipitation with the use of a rabbit polyclonal anti-HAUSP, a combination of anti-Hdmx antibodies p55/p56, and a nonimmune antibody as negative control. Hdmx and HAUSP proteins in immunoprecipitates and extracts were detected with anti-HAUSP monoclonal antibody 1G7 and anti-Hdmx monoclonal antibody 6B1A.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4 HAUSP Directly Deubiquitinates Hdmx
(A) In vitro deubiquitination of Hdmx by HAUSP. In vitro-translated Hdmx was ubiquitinated by GST-Mdm2 in vitro. After stopping the ubiquitination reaction with EDTA, purified wild-type HAUSP or the HAUSP (C223S) mutant was added and incubation resumed.
(B) U2OS cells (6 cm dishes) were transfected with 500 ng of HA-Hdmx, without or with Hdm2 (200 ng), HAUSP (2 μg), or HAUSP (C223S) (2 μg), all in the presence of 1 μg of a His6-tagged ubiquitin expression construct. 24 hr later, cells were treated with MG132 (20 μM) for 6 hr prior to harvesting; the His-purified fraction was analyzed for ubiquitinated Hdmx (anti-HA) and ubiquitinated Hdm2 (4B2), whereas the total lysates were used to detect total levels of Hdmx (anti-HA), Hdm2 (4B2), and HAUSP (1G7).
(C) U2OS cells were transfected as in (B) with indicated constructs and the use 3 μg FLAG-CYLD vector.
(D) U2OS cells were transfected as indicated by using 500 ng HA-Hdmx, 200 ng HA-Hdm2, 3 μg Myc-HAUSP, or 3 μg FLAG-CYLD. Western blot was performed on total lysates. Hdmx and Hdm2 were detected with the anti-HA antibody, HAUSP with anti-Myc antibody, and CYLD with anti-FLAG.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5 DNA Damage Prevents Stabilization of Hdmx by HAUSP
(A) LS89 cells were incubated with doxycycline (1 μg/ml) for 48 hr to induce HAUSP expression. The cells were subsequently mock treated or treated with 500 ng/ml NCS for the indicated time points. Total cell extracts were analyzed as described in Figure 1D. In addition, the anti-phospho-Serine15-p53 antibody was used to monitor the DNA damage response.
(B and C) LS89 cells were incubated with doxycycline (1 μg/ml) for 48 hr to induce HAUSP expression or mock treated. Subsequently, the cells were treated for 2 hr with NCS (500 ng/ml) or mock treated and then incubated with cycloheximide for the indicated time points. Although the levels of Hdm2 and Hdmx were increased by HAUSP induction, exposures were chosen such that the signals at t = 0 are similar to facilitate comparison of the stability.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
DNA Damage Inhibits the Interaction between HAUSP-Hdmx and HAUSP-Hdm2
(A) Transfections were performed into U2OS cells, and cell extracts were analyzed as described for Figure 4B. Cells were treated with MG132 (20 μM) for 5 hr and, where indicated, with 500 ng/ml NCS prior to harvesting.
(B) Immunoprecipitated HAUSP was incubated with the HA-Ub probe and resolved on SDS-PAGE. Active HAUSP was visualized with anti-HA monoclonal antibody, whereas the total amount of immunoprecipitated HAUSP was detected with anti-HAUSP monoclonal antibody.
(C) MCF7 cells were treated with NCS (1000 ng/ml) for 3 hr or mock treated. Immunoprecipitations were performed with the anti-rabbit polyclonal HAUSP antibody, after which the immunoprecipitates and total lysates were analyzed as in Figure 1D.
(D) Hdmx was immunoprecipitated from mock-treated or NCS-treated (1000 ng/ml) cells. Where indicated, the anti-Hdmx immunoprecipitate was treated with alkaline phosphatase in the presence or absence of phosphatase inhibitors (PI). Each immunoprecipitate was subsequently divided and incubated with in vitro-translated p53 (20%) or in vitro-translated HAUSP (70%), after which the bound proteins were analyzed by gel electrophoresis and autoradiography. 10% of each immunoprecipitation was analyzed by immunoblotting to control for the amount of Hdmx immunoprecipitated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7 A Model for HAUSP Function in the Hdmx, Hdm2, p53 Pathway
(A) In normal growing cells, Hdmx stabilizes Hdm2 by inhibition of autoubiquitination, whereas Hdm2 degrades both p53 and Hdmx. The levels of Hdmx, Hdm2, and p53 are regulated by a balance between ubiquitination by Hdm2 and deubiquitination by HAUSP. Therefore, HAUSP impinges directly on p53 but also indirectly via the p53’s negative regulators Hdm2 and Hdmx.
(B) Upon DNA damage, the interactions between HAUSP and Hdmx/Hdm2 are impaired, leading to a decrease of Hdmx/Hdm2 levels. This reduction of Hdmx and Hdm2 likely contributes to activation of p53 after DNA damage.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions