Anti phospho s118 erα

The following antibodies were used at the indicated dilutions: anti-PKD1 (sc-935; 1/500) and anti-α-actinin (1/5000) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-phospho-PKD1 (1/1000), anti-cleaved PARP (1/1000), anti-ERα (1/2000), anti-phospho-S118-ERα (1/2000) and anti-phospho-S167-ERα (1/2000) were from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit IgG (1/2000; Dako, Glostrup, Denmark) and goat anti-mouse IgG (1/5000; Rockland, Gilbertsville, PA, USA). PKD1-targeting (sc-36245) and control non-targeting (sc-37007) siRNAs were purchased from Santa Cruz Biotechnology. 17β-estradiol, ICI 182,780, MTT and all other biochemicals were from Sigma-Aldrich (St. Louis, MO, USA).

17β-estradiol, DMEM (with and without phenol red) and fetal calf serum were purchased from Sigma-Aldrich (St. Louis, MO). The Bradford protein assay kit, as well as anti-mouse and anti-rabbit secondary antibodies, were obtained from Bio-Rad (Hercules, CA). Antibodies against ERα (HC-20 rabbit; F-10 mouse), cyclin D1 (H-295 rabbit), Bcl-2 (C2 mouse), progesterone receptor (C20 rabbit), cathepsin D (H75 rabbit), pS2 (FL-84 rabbit), dynamin II (C-18 goat), p62/SQSTM (D-3 mouse) and anti-goat secondary antibody (sc-2020) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-vinculin, anti-tubulin and anti-LC3 antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Anti-phospho-AKT, anti-AKT, anti-phospho-S118 ERα, and anti-IGF-1R were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-biotin-HRP was purchased from Thermo Fisher Scientific (Walthman, MA, USA). Chemiluminescence reagent for Western blotting was obtained from BioRad Laboratories (Hercules, CA, USA). Dynole 2–24 was purchased from Abcam (USA). All other products were from Sigma-Aldrich. Analytical- or reagent-grade products, without further purification, were used. The identities of all of the cell lines that were used (i.e., human breast carcinoma cells [MCF-7; T47D-1]) were verified by STR analysis (BMR Genomics, Italy).