Expression of Ephb4 was monitored by Western blot analysis. To induce EphB4 expression, doxycycline of various concentrations (0, 500, and 1,000 ng/ml) was added to the mouse ES cell growth medium for 2 days, and then the cells were lysed in RIPA buffer (1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 10 μM leupeptin, 150 mM NaCl, 50 mM Tris, pH 7.4) supplemented with 1% protease inhibitor cocktail (Sigma–Aldrich) [Bai et al., 2012 (link)]. Western blot analyses were performed with anti-EphB4 antibodies (BD Biosciences) as primary antibodies and anti-rabbit IgG-HRP antibodies (Sigma–Aldrich) as secondary antibodies. The protein expression levels were quantified using Photoshop software based on band area and gray scale.