Skip to main content
. Author manuscript; available in PMC: 2010 Jun 11.
Published in final edited form as: Cell Death Differ. 2009 Jul 10;16(11):1493–1504. doi: 10.1038/cdd.2009.89

Figure 4. Disruption of the p53-Siva1-Hdm2 complex by DNA damage signaling.

Figure 4

(A) & (B) Doxorubicin reduces the interaction between Siva1 and p53. (A) H1299 cells were co-transfected with Flag-Siva1 and GFP-p53. After 24 h, cells were treated with 20 μM MG-132 for 2 h and further treated with or without 4 μM doxorubicin for an additional 6 h. The whole cell lysates were prepared for immunoprecipitation with anti-Flag M2 beads, and followed by immunoblotting with anti-Flag and anti-GFP antibodies. (B) A549 cells were treated with 20 μM MG-132 for 2 h, and further treated with or without 4 μM doxorubicin for an additional 6 h, and the whole cell lysates were then immunoprecipitated with anti-p53 antibodies, followed by immunoblotting with anti-p53 and anti-Siva antibodies.

(C) & (D) Doxorubicin reduces the interaction between Siva1 and Hdm2. (C) p53−/−Mdm2−/− MEF cells were co-transfected with Hdm2 and either Flag vector or Flag-Siva1. After 24 h, the cells were treated with 20 μM MG-132 for 2 h, followed by treating with or without 4 μM doxorubicin for an additional 6 h. Whole cell lysates were collected and immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-Flag and anti-Hdm2 antibodies. (D) U2OS cells were treated with 20 μM MG-132 for 2 h, followed by treatment with or without 4 μM doxorubicin for 6 h. Whole cell lysates were immunoprecipitated with either anti-Siva antibody or a control antibody, followed by immunoblotting with anti-Hdm2 and anti-Siva antibodies.

(E) ATM regulates the p53-Hdm2-Siva1 complex. HCT116 cells were transfected with a control siRNA or an ATM-specific siRNA. After 72 h, cells were treated with 20 μM MG-132 for 2 h and with 4 μM doxorubicin for an additional 6 h. The whole cell lysates were prepared for immunoprecipitation with anti-Hdm2 or anti-p53 antibody, and followed by immunoblotting with indicated antibodies (left part). Input (right part) was equivalent to 10% of the whole cell lysate used for Co-IP.