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. Author manuscript; available in PMC: 2013 Jan 27.
Published in final edited form as: Mol Cell. 2011 Dec 22;45(2):171–184. doi: 10.1016/j.molcel.2011.11.018

Figure 2. IKKα-Mediated NOTCH1 Activation Requires NUMB.

Figure 2

(A) Hep3B and Huh-7 cells were treated with TNFα (20 ng/ml) for 24 h. Endogenous NICD and NUMB expression levels were examined by western blotting. α-Tubulin was used as loading control.

(B) Knockdown of IKKα or double knockdown of IKKα and NUMB by siRNA in Hep3B cells. Nontargeting siRNA was used as control. After 24 h of TNFα treatment, cell lysates (30 µg) were subjected to western blotting to detect endogenous NICD and NUMB expression. α-Tubulin was used as loading control.

(C) Western blot analysis of endogenous NICD and NUMB expression in WT MEFs and IKKα−/− MEFs with or without reconstituted IKKα. α-Tubulin was used as loading control.

(D) Hep3B cells were transfected with WT IKKα or KD IKKα. Endogenous NICD and NUMB expression was analyzed by western blotting. α-Tubulin was used as loading control.

(E) RBP-Jk Luc and NUMB were transfected into Hep3B cells for 48 h. After transfection, Hep3B cells were treated with TNFα (20ng/ml) for 24 h. Cell lysates were subjected to the luciferase reporter assay. NUMB expression was analyzed by western blotting with 10 µg of total lysates. Error bars represent SD (n = 3). * indicates statistical significance (P < 0.05).

(F) Hep3B cells were infected with retrovirus expressing IKKα or IKKα and NUMB. RNA extracts were purified from the cell lysates and quantitated with real-time PCR. The NOTCH1 target gene mRNA levels were normalized to the mRNA levels of target genes in vector-infected cells. Error bars represent SD (n = 3). * indicates statistical significance (P < 0.05).

(G) Hep3B and Huh-7 cells were treated with TNFα (20 ng/ml) for 24 h. NUMB mRNA level was detected with qPCR. Error bars represent SD (n = 3). * indicates statistical significance (P < 0.05).

(H) The TRANSFAC 7.0 and PROMO 8.3 programs were used to predict transcriptional factors that bind to the NUMB promoter. Five HNFs were identified from the prediction.

(I) Each of the five indicated HNFs was transfected alone or together with IKKα into Hep3B cells. NUMB mRNA level was detected with real-time PCR. Error bars represent SD (n = 3). * indicates statistical significance (P < 0.05).