Amersham ecl western blotting system

The standard Western blotting protocol (BioRad) was used with the following specifications. The utricles containing sensory epithelia, transitional epithelia, and underlying mesenchyme were isolated by microdissection and lysed in 50 μL RIPA lysis buffer for 30 min at 4 ˚C and sonicated thrice at low power for 10 s each with the sample kept on ice between the sonications. The total protein concentration in each sample was determined by the BCA assay (Thermo Fisher). A NuPAGE 12% Bis-Tris Protein Gel (Thermo Fisher) was used to resolve the proteins in 5 μg of each sample. The proteins were transferred to a nitrocellulose membrane (BioRad) and blocked for 1 hr at room tempirature in a 5% solution of skim-milk powder (Sigma-Aldrich) in tris buffer (BioRad) with 0.1% Tween 20 (Sigma-Aldrich). After the primary antibodies—rabbit anti-Yap (Cell Signaling) and rabbit anti-H3 (Millipore)—had been reconstituted at 1:10000 in tris buffer blocking solution containing 0.1% Tween 20% and 5% normal sheep serum (Sigma-Aldrich), the membrane was incubated over night at 4 ˚C. After 5 30 min washes at room tempirature in TBST, the anti-rabbit HRP secondary antibody (Millipore) was applied in TBST for 1 hr at room temperature. Horseradish-peroxidase activity was detected with the Amersham ECL Western Blotting System (GE Healthcare Life Sciences).