Role of CK2, 14-3-3 and Pin1 in the mechanism of SFN-induced HDAC3 protein loss. (A) Nuclear extracts from SFN-treated HCT116 cells were immunoblotted for phospho-HDAC3 (p-HDAC3), phospho-SMRT (p-SMRT), HDAC3, and SMRT. (B,C) Time-course of 14-3-3, CK2, and Pin1 protein expression changes in cytoplasmic and nuclear extracts of HCT116 cells, normalized to β-actin (not shown). (D) Immunoprecipitation (IP) studies, pulling-down HDAC3 from cytoplasmic and nuclear extracts of HCT116 cells followed by immunoblotting (IB) for SMRT, p-SMRT, 14-3-3, Pin1, and (not shown) HDAC3. (E) siRNA-mediated knockdown of Pin1, compared to scrambled siRNA control. Cells were transfected with siRNAs, 24 h later SFN (15 μM) was added, and whole cell lysates were immunoblotted 16 h thereafter for HDAC3, Pin1, H4K12ac, and p21WAF1.