Anti ambn

The proteins collected from mES cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Boston, MA, USA). After blocking with 5% nonfat milk for 1 h at room temperature, the membrane was incubated for 2 h at room temperature with anti-AMBN (1:500; Santa Cruz Biotechnology), anti-β-catenin (1:500; Santa Cruz Biotechnology), anti-p-Smad1/5/8 (1:500; Santa Cruz Biotechnology), or anti-β-actin (1:1,000; Santa Cruz Biotechnology) antibodies. The blots were subsequently washed with Tween Tris-buffered saline (TTBS; 10 mM Tris–HCl, 50 mM NaCl, and 0.25% Tween) and incubated with the appropriate secondary antibody (Santa Cruz Biotechnology) for 30 min at room temperature.

EO cells were isolated for immunohistochemical analysis and, after 24 h plating on 25 mm coverslips, fixed with 4% paraformaldehyde. Immunofluorescence staining was performed as follows: briefly, the cells were blocked and permeabilized with BSA 1%, Triton 0.1% in PBS for 5 min. After two washes in PBS, the following primary antibodies were used: anti-Ambn (1:500 dilution; SantaCruz sc-50534), and anti-AmelX (1:500 dilution; SantaCruz; sc-32892). After washing, detection was carried out using Alexa Fluor 488 (1:500 dilution; Life Technologies). Samples were embedded using Fluoromount mounting medium (Novus) containing DAPI. Images were taken using a Leica TCS SP5 II confocal microscope and edited using ImageJ (NIH).