4 to 12 bis tris protein gradient gels

Cells were harvested after treatment with simvastatin (0.001, 0.01, 0.1, and 1 μM) and E₂ (10 nM), alone or in combination, at the specified time points. Cells were lysed in a lysis buffer (radioimmunoprecipitation assay buffer, Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich). An equal amount of protein lysates was resolved using 4 to 12% Bis-Tris protein gradient gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific). Membranes were blocked using 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 (TBST, Thermo Fisher Scientific) for 1 h at room temperature and incubated with PCNA (CST, #13110), anti- ER-α (Invitrogen, PA5–16440), pERK1/2 (CST, #4695S), ERK1/2 (CST, #4370), pAKT (Santa Cruz; sc-7985), AKT (Santa Cruz; sc-8312), anti-collagen 1 (Thermo Fisher Scientific, PA5–29569), anti-β-actin (Sigma, A3854), anti-Na,K-ATPase (CST, 23565), or anti-lamin B1 (Invitrogen, PA5–19468) at 4 °C overnight on a rocker. Membranes were washed with TBST, co-incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, Chicago, IL) for 1 h at room temperature and visualized using an Azure Imager c300 (Azure Biosystems, Dublin, CA). Band signals were quantified using the NIH ImageJ software (version 1.52r, USA).