Anti phosphotyrosine

Antigen retrieval was done in the same manner as IHC described above. After pretreatment, sections were blocked with 5% normal goat serum in T‐PBS for 1hr at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in T-PBS: anti-Ephrin-B2 (Santa Cruz, SC1010 or SC19227), anti-Eph-B4 (R&D, AF446 or Santa Cruz, SC5536), anti-vWF (Abcam, ab1713), anti-alpha-actin (Abcam, ab5694), anti-phospho-tyrosine (Abcam, ab10321), anti-phospho-Akt1 (Cell Signaling, #9018), or anti-Akt1 (Cell Signaling, #2967). Sections were then treated with secondary antibodies at room temperature for 1–2 hr using goat anti-rabbit Alexa Fluor 488 (Life Technologies, Grand Island, NY), donkey anti-goat Alexa-Fluor-488 (Life Technologies), or donkey anti-rabbit Alexa-Fluor-568 (Life Technologies). Sections were stained with SlowFade® Gold Antifade Mountant with DAPI (Life Technologies) and coverslip applied. Digital fluorescence images were captured and intensity of immunoreactive signal was measured using Image J software (NIH, Bethesda, Maryland). Intensity of merge signal was determined by applying a color threshold selective for yellow signal.

Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Zeng et al., 2018 (link)). Western blot analyses were performed with anti-MRTF-A (Santa Cruz, sc-32909), anti-phosphoserine (Abcam, ab9332), anti-phosphotyrosine (Abcam, ab10321), anti-c-Abl (Santa Cruz, sc-131), anti-α-SMA (Sigma, A2547), anti-Sp1 (Santa Cruz, sc-14027), and anti-β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.