HIPK2 decreases the stability of Notch1-IC through proteasome-dependent degradation. HIPK2 decreases the stability of Notch1-IC through proteasome-dependent.

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HIPK2 decreases the stability of Notch1-IC through proteasome-dependent degradation. HIPK2 decreases the stability of Notch1-IC through proteasome-dependent degradation. A, endogenous coimmunoprecipitation of RBP-Jk with Notch1-IC in HIPK2+/+ and HIPK2−/− MEFs. B, endogenous coimmunoprecipitation of RBP-Jk with Notch1-IC in HEK293 cells expressing shCon and shHIPK2, and treated with 2 μmol/L adriamycin for 12 hours. C, levels of Notch1-IC in HEK293 cells transfected with shCon and shHIPK2 and treated with 2 μmol/L adriamycin for 12 hours and 100 μmol/L cycloheximide (CHX) for the indicated time were determined by Western blotting. D, levels of Notch1-IC in HIPK2+/+ and HIPK2−/− MEFs treated with 2 μmol/L adriamycin for 12 hours and 100 μmol/L cycloheximide for the indicated time were determined by Western blotting. C and D, we quantified the intensity of each band using a densitometer and plotted relative intensities. Data are expressed as means ± SD from three independent experiments. E, luciferase reporter analysis of the transcriptional activity of Notch1-IC in HIPK2+/+ and HIPK2−/− MEFs treated with 5 μmol/L MG132 for 6 hours. Error bars correspond to pooled data from three independent experiments. F, lysates of HIPK2+/+ and HIPK2−/− MEFs treated with MG132 for 6 hours were analyzed by Western blotting with antibodies against Notch1-IC and β-actin. G, endogenous coimmunoprecipitation of Fbw7 with Notch1-IC in HIPK2+/+ and HIPK2−/− MEFs. Eun-Jung Ann et al. Cancer Res 2016;76:4728-4740 ©2016 by American Association for Cancer Research