Phosphorylation-Dependent Ubiquitination of Cyclin E by the SCFFbw7 Ubiquitin Ligase Deanna M. Koepp, Laura K. Schaefer, Xin Ye, Khandan Keyomarsi, Claire Chu, J. Wade Harper, and Stephen J. Elledge

Supplementary Material

Supplemental Figure 1. Inverse correlation of cyclin E protein and Fbw7 RNA expression in normal and tumor breast cells. A) Northern blot of Fbw7 and B) Western blot analysis of cyclin E in normal, immortalized and tumor cell lines. Normal mortal (N) cell strains from generated from reduction mammoplasty of 2 individuals-lane 1, 81N; lane 2, 76N. Immortalized cell line (I) is depicted in lanes 3 (76NE6). Breast cancer cell lines which are estrogen receptor positive (T, ER +ve) are represented in lanes 4 (MCF-7), 5 (T47D), 6 (BT20T), and 7 (ZR75T). Estrogen Receptor negative (T, ER-ve) breast cancer cell lines are represented in lanes 8 (MDA-MB-157), 9 (MD-MB-231), 10 (MDA-MB-436), 11 (HBL-100) and 12 (Hs-758T). For northern blot analysis 20 g of total RNA from each cell line were analyzed on formaldehyde gels and probed with the full length cDNA to Fbw7 or 36B4 used for equal loading. For western blots 50 g of total cell extract were run on 10% SDS polyacrylamide gels. Proteins were transferred to Immobilon P and blots were incubated with anti-cyclin E and immuno-reactive proteins detected with the ECL reagent (Amersham).

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