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. Author manuscript; available in PMC: 2016 Nov 17.
Published in final edited form as: Oncogene. 2016 May 16;35(48):6166–6176. doi: 10.1038/onc.2016.156

Figure 2. ERα supports C/EBPδ protein stability through inhibition of the FBXW7 pathway.

Figure 2

A) Western blot analysis of C/EBPδ and ERα protein expression in ER+ breast cancer cell lines MCF-7, T47D and Cama-1; ER-, basal MDA-MB-231 cells; and non-tumorigenic, basal MCF-10A cells. SE, LE: short and long exposure, respectively. Tubulin was used as loading control. B) Western analysis (left) of C/EBPδ and ERα protein levels and Quantitative PCR (QPCR) analysis (right) of CEBPD and PGR mRNA levels in MCF-7 cells after nucleofection with siRNAs against non-specific control (-, siNS), C/EBPδ (siCEBPD) or ERα (siESR1) (n=3,* P<0.05 and ** P<0.01 when compared to siNS). C) Western and QPCR analysis of T47D cells as in panel B. D) Western and QPCR analysis of Cama-1 cells as in panel B. E) Western and QPCR analysis of MCF-7 cells treated for 48 h with 1 μM of fulvestrant, tamoxifen, or ethanol (EtOH) as solvent (**P<0.01 when compared to EtOH, n=3). F) Western and QPCR analysis of MCF-7 and T47D cells treated with β-estradiol (E2, 1nM) for the indicated times (*P<0.05, ** P<0.01; n=3). G) Western analysis of C/EBPδ and ERα protein in MCF-7 cells transfected with siRNA against ERα (siESR1) or non-specific control (-) and treated 48 h later with (bortezomib (5 μM) for the indicated times. DMSO was used as solvent control. H) Representative Western analysis (top panels) of MCF-7 cells treated with Fulvestrant (1 μM) or solvent control (EtOH) and 48 h later with puromycin (15 μg/ml) for the indicated times. Tubulin and a non-specific (N.S.) band are shown as loading controls. The bottom panel shows quantification of C/EBPδ protein expression normalized to the non-specific band from three independent experiments (* P<0.05 and ** P<0.01). I) Western analysis of the indicated proteins in whole cell extracts from MCF-7 and T47D cells transfected with siRNA against FBXW7 (siFBXW7), ERα (siESR1), or non-specific control (-).