Anti raf b

The analysis of protein expression by Western blotting was performed as previously described [86 (link)]. The primary antibodies used and their dilutions were the followings: anti-p-Akt1-2-3 (sc-7985-R, Santa Cruz Biotechnology), anti-ERK1 (p44)/ERK2 (p42) (Ref. sc-514302, Total ERK, Santa Cruz Biotechnology), anti-Raf-B (Ref. sc-5284 Santa Cruz Biotechnology) at 1:200 dilution; anti-phosphotyrosine204-ERK1 (p44)/ERK2(p42) (Ref. sc-7383, Santa Cruz Biotechnology) at 1:500 dilution; anti-PARP) (Ref. 9542, Cell Signaling Technology) and anti-β-actin (#MAB1501, Merck-Millipore, Burlington, MA, USA), at 1:5000 dilution overnight at 4 °C. At room temperature, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary anti-rabbit (#MAB201P, Merck-Millipore, Burlington, MA, USA) or anti-mouse antibodies (A4416, Sigma-Aldrich, Milan, Italy), for 2 h. Detection of chemiluminescence signals and densitometric analysis of blots were performed using ImageQuant LAS 4000 (GE Healthcare) and ImageLab software (Bio-Rad), respectively.

17β-estradiol (E₂), 4-OHT, anastrozole, insulin, hydrocortisone, cholera toxin, and DMSO were purchased from Sigma (St. Louis, MO). EGF was purchased from Peprotech (Rocky Hill, NJ), horse serum was from Invitrogen (Carlsbad, CA). TAM pellets (5 mg/pellet, 60-day release) and E₂ pellets (0.36 mg/pellet, 60-day release) were purchased from Innovative Research of America (Sarasota, FL, USA). Matrigel™ Matrix Growth Factor Reduced was purchased from BD Biosciences (Bedford, MA, USA). Anti-Raf-B, anti-p21, anti-Bcl-2, anti-Fibronectin and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BreastDefend® (BD) was supplied by EcoNugenics, Inc. (Santa Rosa, CA, USA) and dissolved in DMSO at a concentration of 25 mg/ml then stored at −20 °C. The chemical composition of BD was previously published [26 (link)]. All other chemicals and reagents were of analytical grade.