Anti cyclind

MDA-MB-435 or PC3 cells were treated with vehicle, or 4 or 8 μM Ehop-016 for 24 h. Cells were immediately lysed as in [57] (link) and total protein was quantified using the Precision Red protein assay kit (Cytoskeleton, Inc., Denver, CO). Equal total protein amounts were Western blotted using anti-Akt, anti-phospho Akt^Thr308, anti-JNK, anti-phospho JNK^{Thr183/Try185}, anti-c-Myc, or anti-Cyclin D (Cell Signaling Technology, Inc., Danvers, MA) antibodies. The integrated density of positive bands was quantified using Image J software.

HUVECs were washed twice with PBS, lysed in RIPA buffer [1 mM EDTA, 150 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid, pH 7.4], and heated at 98°C for 10 min. The protein concentration was measured using the bicinchoninic acid method. Equal amounts of protein (20 µg protein/well) were separated using a 9% SDS/polyacrylamide gel and transferred onto a PVDF (EMD Millipore) membrane at 100 V for 2 h at 4°C. The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies, including anti-β-catenin (cat. no. 8480S; 1:1,000; Cell Signaling Technology), anti-Cyclin D (cat. no. ab16663; 1:100; Abcam), anti-IL-6 (cat. no. sc-57135; 1:200; Santa Cruz Biotechnology, Inc.) and anti-TNF-α (cat. no. sc-52746; 1:500; Santa Cruz Biotechnology, Inc.), anti-GAPDH (cat. no. ab181602; 1:10,000; Abcam), overnight at 4°C. After washing with TBS-Tween 20 (0.05%), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody anti-Immunoglobulin G (1:2,000; cat. no. A7539; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Finally, the proteins were visualized using an ECL detection system (EMD Millipore) and quantified by densitometry using Quantity One software (version v4.6.6; Bio-Rad Laboratories, Inc.). GAPDH was used for the internal control.

Total cell lysates were prepared in 5× sample loading buffer (250 mM tris hydrochloride [pH 6.8], 40% glycerol [80%, v/v], 8% sodium dodecyl sulfate [SDS], 2% β-mercaptoethanol, 0.1% bromophenol blue, 100 mM dithiothreitol [1 M]. The bicinchoninic acid (BCA) method and BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) were used to quantify the protein concentration of the samples. Equal amounts of protein (20-30 µg) were separated by 6-13% SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), and blocked with 5% bovine serum albumin (Sigma-Aldrich). The membranes were then probed with anti-ACTN-4, anti-α-tubulin, anti-β-Actin, anti-vinculin, anti-FAK, anti-Rac, anti-Slug, anti-Snail, anti-CDK2, anti-CDK4, anti-cyclin E, anti-cyclin D, anti-Bax, anti-Bcl-2, anti-cleaved caspase 3, anti-caspase 3, anti-MMP2, and anti-cytochrome c antibodies purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-N-cadherin antibodies purchased from BD Biosciences overnight with primary antibodies at 4°C. Following the incubation, the membranes were washed three times and further incubated with peroxidase-labeled secondary antibodies for 4 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific).