Quantitative Western Blot Analysis

Total proteins were isolated using an ice-cold Pierce IP lysis buffer (Fisher Scientific, Vienna, Austria) supplemented with complete protease and a phosphatase inhibitor cocktail (Fisher Scientific, Vienna, Austria) [36 (link),38 (link)]. The protein concentration was determined by a Bradford assay (Bio-Rad Laboratories, Vienna, Austria). Equal amounts of protein were incubated in Roti-Load1 Buffer (Carl Roth, Karlsruhe, Germany) at 100 °C for 10 min and then separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary rabbit anti-β-catenin (1:1000, #8480, Cell Signaling, Frankfurt am Main, Germany) or rabbit anti-GAPDH (1:1000, #2118, Cell Signaling, Frankfurt am Main, Germany) antibodies at 4 °C overnight and with a secondary anti-rabbit HRP-conjugated antibody (1:1000, Cell Signaling, Frankfurt am Main, Germany) at RT for 1 h. The membranes were stripped in a stripping buffer (Fisher Scientific, Vienna, Austria) at RT. Bands were detected with an ECL detection reagent (Fisher Scientific, Vienna, Austria) and quantified using ImageJ software ((NIH, MD, USA, 1.52n). The data were shown as the ratio of the total protein to GAPDH, normalized to the control group [38 (link),39 (link)].