Azure 300

MCF-7 and 4T1 cells were seeded in 10 cm plates at a starting density of 10⁶ cells/plate, were cultured overnight, and then were treated with 0, 5, or 10 µM DEA for 24 h. The cells were harvested in a chilled lysis buffer containing 0.5 mM sodium–metavanadate, 1 mM ethylenediamine–tetraacetic acid, and protease and phosphatase inhibitor cocktails (1:200). After boiling, the cell lysates were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis; then, the proteins were transferred to nitrocellulose membranes. After blocking the membranes in 5% bovine serum albumin (BSA) for 1.5 h at room temperature, they were exposed to primary antibodies diluted in blocking solution at 4 °C overnight. Appropriate horseradish peroxidase-conjugated secondary antibodies were used at a dilution of 1:5000 (anti-mouse and anti-rabbit IgGs; Sigma-Aldrich, St. Louis, MI, USA). Chemiluminescence generated by applying the WesternBright ECL HRP substrate (Advansta, San Jose, CA, USA) was measured using an Azure 300 (Azure Biosystems, Dublin, CA, USA) high-resolution imaging system. Pixel volumes of the bands were determined using ImageJ software. For membrane stripping and re-probing, the membranes were washed in a stripping buffer containing 0.1 M glycine and 5 M MgCl₂ (pH 2.8) for 30 min at room temperature. After washing and blocking, the membranes were re-probed.

For WB analysis, total cells were detached by scraping, collected by centrifugation, and lysed in RIPA buffer [50 mMTris·HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate (NaDoc), 0.1% SDS] containing proteases and phosphatase inhibitors (Roche). Protein concentrations were determined using the DC Protein assay (Bio-Rad Laboratories). Cell lysates were resolved on MiniPROTEAN TGX gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories), followed by WB using indicated primary Abs and revealed by using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Bio-Rad Laboratories). Proteins were visualized by chemiluminescence (Clarity Western ECL Substrates, Bio-Rad Laboratories) by using Azure 300 (Azure Biosystems) or by ChemiDoc Imaging System and Image Lab Software (Biorad Laboratories). Quantification analyses were performed using ImageJ and reflected the relative amounts as a ratio of each protein band relative to the loading control of the lane.

B16F10 cells were seeded in 10 cm plates at 10⁶ cells/plate and treated as described above for the cell viability assay. The cells were harvested at intervals in chilled lysis buffer containing 0.5 mM of sodium-metavanadate, 1 mM of EDTA, and protease inhibitor cocktail (1:200). The cell lysates were boiled and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis before being transferred to nitrocellulose membranes. The membranes were blocked in 5% low-fat milk for 1.5 h at room temperature, and then exposed to primary antibodies at 4 °C overnight in blocking solution. Appropriate horseradish peroxidase–conjugated secondary antibodies were used at a dilution of 1:5000. Signals were visualized by using enhanced chemiluminescence and captured on X-ray film. The films were scanned, and the pixel densities of the bands were determined using the NIH ImageJ software. Alternatively, chemiluminescence was measured on an Azure 300 (Azure Biosystems) imaging system that digitized the bands’ chemiluminescence intensities using its inbuilt software. For stripping and reprobing, the membranes were washed in stripping buffer (0.1 M glycine, 5 M MgCl₂, pH 2.8) for 1 h at room temperature. After washing and blocking, the membranes were incubated with primary antibodies against non-phosphorylated or loading control proteins. These experiments were repeated three times.

For WB analysis, cells were detached by scraping, collected by centrifugation, and lysed in RIPA buffer [50 mMTris·HCL (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate (NaDoc), 0.1% SDS] containing proteases and phosphatase inhibitors (Roche). Protein concentrations were determined using the DC Protein assay (Bio-Rad Laboratories). Cell lysates were resolved on MiniPROTEAN TGX gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories), followed by WB using the primary antibodies. Primary antibodies were revealed using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Bio-Rad Laboratories). Proteins were visualized by chemiluminescence (Clarity Western ECL Substrates, Bio-Rad Laboratories) by using Azure 300 (Azure Biosystems). Quantification analyses were performed by ImageJ (https://imagej.nih.gov/ij/), a Java-based freeware, and reflects the relative amounts as a ratio of each protein band relative to the lane’s loading control.