Total p27

Primary antibodies against phospho-src^tyr416, phospho-src^tyr527, src, phospho-ERK1/2 (p42/44), phospho-p90^rsk, phospho-AKT^ser473, total-AKT, cyclin D1, phospho-EGFR^tyr1068, total-EGFR and total p27 were purchased from Cell Signalling; total-ER and phospho-p27^ser10 from Santa Cruz; total ERK1/2 and Actin (Sigma Aldrich) and PgR (Novacastra). Total-ERBB2 and phospho-ERBB2^Tyr1248 were purchased from Millipore. Secondary horseradish peroxidase antibodies were obtained from Dako. 17-β-estradiol (E2) and 4-hydroxytamoxifen (4-OHT) were purchased from Sigma-Aldrich, UK; fulvestrant (ICI182780) from Tocris Bioscience and dasatinib from Selleckchem.

Proteins were extracted in RIPA buffer and concentrations were measured by the bicinchoninic acid method (Pierce, IL). Total extracts were electrophoretically fractionated on 10% or 12% Bis-Tris polyacrylamide gels (Invitrogen) and transferred to a 0.45 μm nitrocellulose membrane. Membranes were blocked for 1 h with 5% nonfat dry milk in TBS-Tween and incubated overnight at 4°C with the following primary antibodies: phospho-p38, phospho-Akt, phospho-Erk1/2, and total p27, p38, Akt and ERK (diluted 1:1000, Cell Signaling, Danvers, MA), p21 (Dako, 1:1000), and β-actin (Sigma, 1:5000). Membranes were washed and incubated with peroxidase-labeled secondary antibodies at room temperature for 1 h. Immunoreactive bands were detected with the Lumi-Light Western Blotting Kit (Roche, Switzerland) and quantified with ImageJ.