17β estrogen e2

Rabbit anti-Myc, anti-Flag, mouse anti-Flag (M2) and cycloheximide (CHX) were obtained from Sigma. Rabbit GFP was purchased from GeneTex. Rabbit anti-PES1and 17β-estrogen (E2) were obtained from Abcam. Rabbit anti-ERα was obtained from Millipore. MG132 was purchased from Merk. The siRNA sequences, siTrim23 and si control, were obtained from GenePharma RNAi Company and their sequences are shown in Supplementary Table S1.
HA-PES1 was obtained from Dr. Dirk Eick (Institute of Clinical Molecular Biology and Tumor Genetics, GSF Research Center, Germany). GFP-PES1 was constructed in our laboratory. The PES1 mutants GFP-K249R, K517R, and 2KR were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using HA-PES1 as the template. Myc-tagged SUMO-1, SUMO-2, SUMO-3 along with GFP-SUMO1 and its mutant GFP-SUMO1/GA have been used in our previous studies [11 (link), 12 (link)]. Flag-UBC9 was amplified by PCR and subcloned into Flag-pCDNA3.1 vector at Xho1 and EcoR1 sites.

Human breast cancer cell lines MCF-7, T47D, MCF10A and MDA-MB-231 have been used in our previous studies [31] –[34] (link). MCF-7 and MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (Hyclone, Logan, UT), 100 µg/ml penicillin and 100 µg/ml streptomycin. T47D cells were cultured as previously described [35] (link). MCF10A were cultured in DMEM/F12 (1∶1) containing 5% horse serum (Hyclone, Logan, UT), 100 ng/ml Cholera Toxin, 10 µg/ml bovine insulin, 0.5 µg/ml hydrocortisones (sigma) and 20 ng/ml EGF (Peprotech, Rehovot Israel). Unless otherwise stated, all cell cultures were incubated at 37°C in the presence of 5% CO₂. Cycloheximide (CHX), actinomycin D (Act D) and anti-FLAG antibody were obtained from Sigma. Anti-ERα, anti-HA, and anti-CLOCK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and 17β-estrogen (E2) and ICI 182780 (ICI) were obtained from Abcam (Cambridge, UK).