Tropix cdp star

Isolated CECs were lysed with RIPA buffer (150 mM sodium chloride, 1% Triton X-100, 0.1% SDS, 50 mM Tris, pH 8.0) containing protease inhibitor cocktail (Sigma). The proteins were separated by 10%–15% SDS–PAGE and blotted onto a PVDF membrane (Bio-Rad). Membranes were blocked in TBS containing 0.1% Tween^® 20 (Fisher BioReagents) and 5% skim milk powder (Oxoid) for 1 h at room temperature. Membranes were incubated with total β-catenin (H-102) (Santa Cruz Biotechnology), non-phospho (active) β-catenin (Ser33/37/Thr41), p-53 (Ser15) and EphB2 (D2X2I) obtained from Cell Signaling, or p-GSK-3α/β (Tyr279/216), p21 (F-5), and DKK-1 (B-7) from Santa Cruz Biotechnology. β-actin (Cell Signaling), GAPDH (Sigma), or β-tubulin (Thermo Fisher Scientific) was used as loading control. Typically, the primary antibodies were used at 1:1000 at 4°C overnight incubation. After washing three times with TBST, the membranes were incubated with goat anti-rabbit IgG-AP (Santa Cruz Biotechnology) or goat anti-mouse IgG-AP (Cell Signaling) at 1:20000 for 1 h at room temperature. Protein–antibody complexes were visualized using a chemiluminescent substrate Tropix^® CDP-Star^® (Applied Biosystems) and detected on CL-X Posure™ Film (Thermo Scientific). Each western blot was repeated at least three times, and the protein expression levels were determined by densitometry analysis and ImageJ.