Nucleic Acids Research, 2009, Vol. 37, No. 9 3115 (Figure 4A and B). Moreover, overexpression of CK1d in MCF7 cells resulted in stabilization of AIB1 (data not shown). Since AIB1 can be degraded through the 26S protea- some (as well as the REGg-mediated proteasome path- way) we examined whether AIB1 downregulation could be rescued by MG132. Use of MG132 rescued the CK1d siRNA-mediated decrease of AIB1 in E2-treated MCF7 cells (Figure 4C and D) (similar results were obtained using another independent CK1d siRNA). These results imply an involvement of CK1d in the proteasome- dependent regulation of AIB1 expression. Figure 2. CK1d does not phosphorylate ERa at S118. (A) MCF7 cells (2 105) were plated in 6-well plates in phenol red-free DMEM containing 10% DSS. Cells were untransfected or transfected with 5 nM CT siRNA or with 5 nM CK1d siRNA for 72 h and treated or not with E2 (100 nM) for 45 min. Cells were harvested, lysed and equal protein amounts were subjected to Western blotting analysis using the indicated antibodies. b-actin was used as a control for sample loading. Immunoblot showing silencing of CK1d. (B) Quantitative analysis of ERa pS118, ERa protein levels and the pS118:ERa ratio is given as fold of control. (C) Western blotting of the levels of ERa expressed in the stably transfected Hela-ERa cell line. (D) Hela-ERa cells untransfected or transfected with 5 nM CT siRNA or with 5 nM CK1d siRNA for 72 h were treated or not with E2 (100 nM) for 45 min. Cell extracts were immunoblotted. (E) Quantitative analysis of ERa phospho-S118, is given as fold of control. All data are representative of results from two independent experiments. Error bars represent SD of two separate experiments in triplicate. described in ‘Materials and Methods’ section. Gene expression of (C) pS2 and (D) PR was measured by quantitative real-time RT–PCR. MCF-7 cells (2 105) were plated in 6-well plates in phenol red-free DMEM containing 10% DSS. Following, cells were incubated for 24 h with 1mM of the CK1d/e-speciﬁc inhibitor IC261 and treated or not (vehicle) with E2 (10 nM) for 24 h. Cells were harvested and total RNA was extracted and used to synthesize cDNA by reverse transcription, as described in ‘Materials and Methods’ section. Gene expression of (E) pS2 and, (F) PR was measured by quantitative real-time RT–PCR. Transfection of MCF7 cells with a (EYFP)-CK1d plasmid resulted in an increase of (G) pS2 expression. GAPDH was used for normalisation. Error bars represent SD of two separate experiments, each in triplicate; changes observed throughout these experiments were statistically signiﬁcant (P < 0.05).