Western Blot Analysis of Cellular Proteins

Cells were seeded in 75 cm² bottles and after 24 h incubation to attach and stretch, treated with the different compounds. After 40 h cells were washed with cold HANK’s balanced salt solution and lysed and scraped with RIPA buffer [5 (link)]. After 20 min incubation on ice, samples were centrifuged for 15 min at 16,000 g and 4 °C. Protein concentration was determined using Bio-Rad Dc Protein Assay (Bio-Rad Laboratories). Twenty micrograms of protein from total cell lysates was subjected to SDS-PAGE and analyzed by Western blot. Primary antibodies used in this study were directed against β-Catenin (Ab6302 1:4000) (Abcam, Cambridge, UK), HER2 (PA5-14635 1:500) (Pierce-Thermo Scientific), HER3 (PA1-86644 1:2500 (Thermo Scientific), with β-Actin pan Ab-5 (MS-1295-P1 1:2000) (Thermo Scientific) as a reference protein. And as secondary antibody, goat anti-mouse HRP-conjugated (HAF007), goat anti-rabbit HRP conjugated (HAF008) and donkey anti-goat HRP conjugated (HAF109) (R&D Systems, Abingdon, UK) was used in a 1:20.000 dilution. HRP was visualized using Advance TM_Enhanced chemiluminescence (ECL, Amersham, GE Healthcare, Eindhoven, The Netherlands) and analyzed using GelDoc 2000 (Bio Rad). With the Quantity One software, version 4.6.9 (BioRad), densities were measured, corrected for the background and related to β-Actin expression as loading control.