Western blot analyses

Cell lysates were mixed with LDS sample buffer supplemented with sample reducing agent (invitrogen) and boiled for 5 min at 95°C. 10–15 mg of protein/lane was run through 10% Bis–Tris gels (120 V, 90 min). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes with a Bio‐Rad Mini Trans‐Blot System (35 V, 3 h). Membranes were blocked with 5% milk/PBS‐T (phosphate‐buffered saline supplemented with tween 20) for 45 min in room temperature (RT) and incubated with primary antibodies diluted in 5% bovine serum albumin/PBS‐T overnight at 4°C. Blots were washed with PBS‐T (four times, 15 min/wash) and incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (1 h, RT). After four washes with 15 min in PBS‐T, blots were incubated in WesternBright Quantum (Advansta) and imaged by using a Fusion SL imaging System (Vilmer). The following primary antibodies were used for Western blot: mouse anti‐a‐tubulin (T6074, Sigma‐Aldrich), rabbit anti‐TCF7 (C63D9, Cell Signaling), rabbit anti‐LEF1 (C12A5, Cell Signaling), rabbit anti‐TCF7L1 (D15G11, Cell Signaling), rabbit anti‐TCF7L2 (C48H11, Cell Signaling), rabbit anti‐β‐catenin (SantaCruz sc‐7199). Horseradish peroxidase‐conjugated secondary anti‐mouse and anti‐rabbit antibodies for Western blotting were purchased from Jackson.