Sds page minigels

Protein extracts were prepared as previously published by our laboratory; cells were homogenized on ice in RIPA buffer (50 mM Tris, pH 8.0 containing 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, and 1% NP40) containing protease inhibitors. Protein concentrations were measured using the Bradford method. Samples were mixed with loading buffer containing β-mercaptoethanol and boiled for 5 min. One-hundred micrograms of each sample were then separated in SDS-PAGE mini gels (BioRad) and transferred to PVDF membranes (Amersham Biosciences, Uppsala, Sweden). The membranes were incubated for 1h at room temperature in blocking buffer (5% fat free milk, 0.1% Tween-20 in PBS). Primary antibodies were used at a 1/200–1/2000 dilution in blocking buffer and were incubated at 4 °C overnight. After washing with PBST, membranes were incubated with secondary antibodies at a 1/1000 dilution for 1 h at room temperature. Signals were detected with an enhanced chemiluminescence kit (ECL, Amersham Biosciences). Primary antibodies: rabbit anti-ER alpha, rabbit anti-E-cadherin, rabbit PR (Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibody: donkey-anti-rabbit HRP.

HEK293 cells (3 × 10⁵) were seeded in 6-well plate and treatment with selected extracts was performed for 16 h unless indicated. For western blot analysis, cells were collected and homogenized in 1% Triton X-100 in PBS containing proteases and phosphatases inhibitors (Roche). After sonication, protein concentration was determined in all experiments by micro-BCA assay (Pierce), and 10–30 µg of total protein was loaded in 8–15% SDS-PAGE minigels (Bio-Rad Laboratories, Hercules, CA) prior transfer into PVDF membranes. Membranes were blocked using PBS, 0.1% Tween-20 (PBST) containing 5% milk for 60 min at room temperature and then probed overnight with primary antibodies in PBS, 0.02% Tween-20 (PBST) containing 5% skimmed milk. The following primary antibodies and dilutions were used: anti-GFP 1:1,000 (Santa Cruz, Cat. n° SC-9996), anti-HSP90 1:5,000 (Santa Cruz, Cat. n° SC-13119), anti-LC3 1:1,000 (Cell signaling, Cat. n° 3868), anti-p62 1:5,000 (Abcam, Cat. n° ab56416), Anti-pCHK1(Ser345) 1:1,000 (Cell Signaling, Cat. n° CST-2348). Bound antibodies were detected with peroxidase-coupled secondary antibodies incubated for 2 h at room temperature and the ECL system.