4.8. Western Blot Protein Analysis

Cells were lysed in either RIPA buffer (Millipore, Burlington, MA, USA) for total protein extracts or with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher,) for nuclear and cytoplasmic protein fractions after 24 h of treatment with inhibitors or vehicle control. Equal amounts of protein (15–20 µg) were denatured, separated on SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene-difluoride-membranes (Millipore). After blocking in 5% skim milk (AppliChem)/TBS-T for 30 min, membranes were probed with primary antibody in TBS-T over night at 4 °C. Following secondary antibody incubation, proteins were visualized with chemiluminescent substrate (ECL prime Western Blotting Detection Reagent, Sigma-Aldrich). The following primary antibodies were used: AXIN1 (Cell Signalling Technology, Danvers, MA, USA, [CS]#2087), non-phospho (active)-β-catenin (CS#8814), YAP (Santa Cruz Biotechnology, Santa Cruz CA, USA, sc-101199), P-YAP (ser127) (CS#4911), FOXM1 (Santa Cruz Biotechnology, sc-271746), total β-catenin (BD Transduction Laboratories, BD Biosciences, CA, USA, 610153), Tankyrase 1/2 (E10) (Santa Cruz Biotechnology, sc-365897), MEK1/2 (CS#9122), P-MEK1/2 (ser217/221) (CS#9121), GAPDH (Santa Cruz Biotechnology, sc-32233), LAMIN B1 (ab16048-100, Abcam,), ACTIN (A2066, Sigma). Primary antibodies were visualized with secondary IgG-HRP conjugated antibodies (715-035-150 [mouse] or 711-035-152 [rabbit], Jackson ImmunoResearch, West Grove, PA, USA) and enhanced with chemiluminescent substrate (ECL prime Western Blotting Detection Reagent, Sigma-Aldrich, RPN2236). Figures show representative data. ACTIN, GAPDH or LAMINB1 documents equal protein loading. Loading controls were always run on the same blot used for the experimental samples.