Mouse anti dvl3

The hippocampus and cortex of treated and control mice were dissected on ice and either immediately processed or frozen at −150°C. Immunoblotting was performed as previously described (Varela-Nallar et al., 2009 (link)). Primary antibodies used were: mouse anti-Dvl3, mouse anti-β-catenin, mouse anti-c-myc, mouse anti-cyclin D1 and rabbit anti-β-tubulin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-rabbit anti-HIF-1α (Novus Biologicals, Littleton, CO, USA).

Cells and biopsies were lysed with a buffer containing 1% Triton X‐l00, 50 mM HEPES (pH 7.5), 150 mM NaC1, 10% glycerol, 1.5 mM MgCl2, 5 mM EGTA, protease inhibitors (4 mM phenyl methylsulfonyl fluoride and 100 mg/ml aprotinin; Sigma‐Aldrich), phosphatase inhibitors (10 mM sodium orthovanadate and 20 mM sodium pyrophosphate; Sigma‐Aldrich) and processed. For direct immunoblot analysis, we employed 20 μg of total cellular proteins, re‐suspended with 25 μl of loading buffer, boiled for 5 minutes and loaded on SDS‐PAGE for Western blot (WB). The antibodies for WB were used at the condition suggested by the suppliers: mouse anti‐Dvl3 (sc‐271295, 1/500; Santa Cruz Biotechnology, Texas, USA), mouse anti‐Yap1 (sc‐376830, 1/500; Santa Cruz Biotechnology), mouse anti‐Smad1/2/3 (sc‐7960, 1/500; Santa Cruz Biotechnology), rabbit anti‐cyclin D1 (E3P5S #55506, 1/1000; Cell Signaling) and mouse anti‐beta‐actin (sc‐81178, 1/1000; Santa Cruz Biotechnology). The WB has been acquired by the ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., California, USA), and the corresponding bands have been quantified by Image lab 6.1.0 (Bio‐Rad Laboratories, Inc.).