Ecl advance reagent

Western blotting was performed as previously described [15 (link)]. Quantified protein lysates (20 μg) were resolved in an SDS-PAGE gel and were transferred onto a PVDF membrane. The membrane was then blocked in 5% bovine serum albumin (BSA) for 2 h, washed with TBS and incubated with the primary antibody overnight at 4°C. After incubation with the secondary antibody for 2 h at room temperature, the immunoreaction was visualized using the ECL Advance reagent (GE Healthcare) and was quantified using Quantity One software.

To determine the protein concentration, cells were lysed in RIPA buffer (Sigma, St. Louis, MO) containing a protease inhibitor mixture (Roche Diagnostics, IN). The protein concentration was measured with the bicinchoninic acid assay (Thermo Fisher Scientific, Bonn, Germany). Quantified protein lysates (15 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and resolved on 12% polyacrylamide gels. The proteins were then transferred onto a PVDF membrane (GE Healthcare, Freiburg, Germany). The membrane was probed with primary antibodies, including WIF1, β-catenin, P-β-catenin (Ser33/37/Thr41), Akt, P-Akt (Ser473), GSK3β, P-GSK3β (Ser9), DNMT1, DNMT3b, lamin B and β-actin (Cell Signaling Technology, Danvers, MA) at 4°C overnight. After three washes with Tris-buffered saline with 0.05% Tween-20, the membrane was incubated with secondary anti-rabbit or anti-mouse antibodies (Cell Signaling Technology, Danvers, MA) for 2 h at room temperature. The signals were visualized using the ECL Advance reagent (GE Healthcare) and quantified using Quantity One software.

Dodecapeptides spanning the whole sequence of RepA-WH1, with an overlap of ten residues (Fig. 2c), were solid-phase synthesized (Fmoc) and immobilized (≈20 nmol per spot) in an Amino-PEG₅₀₀-UC540 sheet at the Proteomics facility of the National Centre for Biotechnology (CNB-CSIC, Madrid), as described57 (link). To enhance solvation of hydrophobic peptides, the membranes were briefly rinsed with ethanol. Membranes were washed three times with TBS and incubated with 5% blocking solution (ECL Advance blocking agent; GE Healthcare) in TBS-T for 4 h. Membranes were then probed overnight with primary antibodies (B3H7 or α-WH1, 4.4 ng) and washed three times with TBS-T. The secondary antibodies (HRP-conjugated anti-mouse/rabbit; 1:10,000) were incubated for 2 h, and then blots were washed three times with TBS-T and developed with the ECL Advance reagent (GE Healthcare). For membrane stripping, blots were sequentially incubated with 8 M urea, 1% SDS, 0.5% β-mercaptoethanol in PBS for 30 min at 55 °C and three times with acetic acid/ethanol/Milli-Q water (10:50:40). Membranes were then washed with TBS and incubated with blocking solution before re-hybridization. For each spot, both the antibody signal and peptide load (Ponceau red staining) were quantitated as indicated above for the purified antibodies.