Supplementary Figure 6: Molecular analyses of cyclin C function.
(a,b) Characterization of anti-phospho-Ser2517 ICN1 antibody. (a) Myc-taggedwild-type ICN1 (WT), or the indicated ICN1 mutants (TSS denotes T2512/S2514A/S2517A triple-mutant), or empty vectors (EV) were transfected into 293T cells. Cell lysates were probed with an anti-phospho-ICN1 antibody. (b) Upper panel: wild-type ICN1 (WT), or the indicated ICN1 mutants were expressed in 293T cells, immunoprecipitated with anti-Notch1 antibody, and immunoblotted with anti-phospho-ICN1 antibody. Lower panel: Notch1 was immunoprecipitated from I22 cells [a murine T-ALL line derived from a tumor induced by retrovirally-driven ICN1 (Pear et al. J. Exp. Med. 1996;183:2283)]. Immunoprecipitates were treated with lambda phosphatase (+) or left untreated (−), immunoblotted and probed with an anti-phosho-ICN1 antibody. Phosphatase treatment abolished immunoreactivity, as expected. (c) MOLT-16 cells were transduced with anti-cyclin C or control shRNA (CTR). ICN1 was immunoprecipitated (IP); normal IgG IP was used as a negative control. Immunoblots were probed with the indicated antibodies. Note reduced phosphorylation of endogenous ICN1, and reduced interaction of endogenous ICN1 and Fbw7 proteins in cyclin C-depleted cells. Input, immunoblotting of straight lysates. (d) Cyclin CF/F MEFs were transduced with Myc-tagged-ICN1. Cells were then treated with control adenovirus (CF/F) or with an adenovirus encoding Cre (CΔ/Δ). ICN1-Myc was immunoprecipitated and immunoblots were probed using an antibody against Fbw7, or against Myc. Input, whole cell lysates. Note that the interaction between ICN1 and endogenous Fbw7 was strongly decreased on ablation of cyclin C. (e) HeLa cells were transfected with HA-tagged Fbw7 along with an empty vector (EV), or Myc-tagged wild-type ICN1 (WT), or various ICN mutants containing alanine-substitutions within critical cyclin C-CDK-dependent phosphosites(TSS denotes T2512/S2514A/S2517A triple-mutant). ICN1 was immunoprecipitated using an anti-Myc antibody, and immunoblots were probed with an anti-HA antibody (to detect ICN1-bound Fbw7). Input, whole cell extracts. Note that alanine-substitutions inhibited the interaction of ICN1 with Fbw7 in vivo. (f) Cyclin CF/F MEF were transduced with Notch1 (WT-Notch1) or a Notch1 mutant (TSS-Notch1) in which the three critical cyclin C-CDK phosphorylation sites have been mutated to alanines (T2512A, S2514A S2517A). Cells were then transduced with control adenovirus (CF/F), or an adenovirus encoding Cre (CΔ/Δ). The levels of ICN1 were determined by western blotting. The middle portion of the gel was cut out and the images spliced together (dashed line). The expression of alanine-substituted mutant was strongly, as compared to the wild-type protein, and it was no longer affected by cyclin C-ablation. (g) HEK293 cells were transduced with anti-cyclin C or control shRNA (CTR). Cells were then transfected with Myc-tagged-ICN1 and His-tagged-ubiquitin. Ubiquitinated proteins were immunoprecipitated using Ni-NTA matrices and immunoblots were probed with an anti-Myc antibody to detect ubiquitinated ICN1. Input, whole cell lysates. Densitometric scanning of ubiquitinated ICN1 band intensities (normalized against input Myc-ICN1) revealed approximately 3-fold lower ubiquitination of ICN1 in cells depleted of cyclin C.
