Research Paper Volume 9, Issue 10 pp 2137—2162

Figure 7. SOCS1 favors p53 accumulation in response to Doxorubicin. (A) Western blots of SOCS1, phosphorylated p53 at serine 15 [p-p53 (S15)] total p53 and tubulin in IMR90 cells expressing either empty vector (V) or SOCS1 and treated with doxorubicin (Doxo: 300 ng/mL) for 3, 6, 9, 16 hours or untreated (-). (B) Graphic representation of Western blots as in (A). Bands were quantified using image analysis software and normalized to tubulin, then plotted in a graph to show the kinetics of p53 stabilization. (C) Co-Immunoprecipitation of KAP1 with SOCS1. U2OS cell lysates of either empty vector cells (V) or SOCS1 overexpressing cells (S1) were immunoprecipitated with an antibody against KAP1 or a control antibody (IP ctl). Western blots against both KAP1 and SOCS1 were performed to confirm the presence of SOCS1 in complex with KAP1. Whole cell lysates (WCL) are used to control the expression of SOCS1 and KAP1 levels. (D) Co-immunoprecipitation as described in C. Cell lysates were immunoprecipitated with an antibody against SOCS1 or with a control antibody (IP ctl). Whole cell lysates (WCL) show the expression level of SOCS1 and KAP1. (E) Maps of the different KAP1 constructs used in experiments are depicted: KAP1 full length (KAP1), KAP1 with a deletion of its N-terminal RBCC domains (KAP1 ΔN) or KAP1 C-terminus including PHD and Bromo domains (KAP1 PHD/BROMO). (F) The constructs depicted in E. were expressed by IPTG induction in BL21 bacterial cells. Expression levels of the various constructs were assessed by migration of an SDS-PAGE gel and Coomassie staining. (G) GST pull down was performed on KAP1 constructs which were incubated with radiolabeled SOCS1. Autoradiography revealed the absence or presence of SOCS1 in each pull down. GST was used as a negative control. (H) Model for p53 activation by SOCS1 via two pathways.