Anti vangl2

Harvested cell pellets were lysed for 15 min with ice-cold lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM sodium chloride, 0.1 % SDS, 1 mM EDTA, 1× Roche protease inhibitor cocktail). For Western blot analyses, samples containing equal amounts of protein were separated by gel electrophoresis and electroblotted onto Hybond-P membranes (Amersham Pharmacia, Cleveland, OH USA). Blots were blocked with 5 % skim milk, followed by incubation with antibodies specific for anti-Vangl2 (1:1000, R&D Systems, Minneapolis, MN USA), anti-Prickle1 (1:1000; Santa Cruz Biotechnology), anti-full length β-catenin (1:1000, Cell Signaling Technology), anti-active β-catenin (1:1000, clone 8E7, Millipore, Solna, Sweden), anti-Axin2 (1:1000, Cell Signaling Technology), anti-β-actin (1:5000, Cell Signaling Technology) and anti-GADPH (1:10000, Millipore). Blots were further incubated with goat anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham) accordingly to manufactures instruction and developed on Kodak hyperfilm. Quantification of blots were done with densitometry measurements in ImageJ [15 (link)].

Dissected otic capsules from mice at P1 were fixed in 4% paraformaldehyde at 4 °C for 2 h. Then, the cochleae were dissected from the otic capsules and immunohistochemically analyzed as previously described [26 (link)]. Unless otherwise noted, all imaging pictures were taken from the mid-basal region of the cochlea. The following primary antibodies were used: anti-Dmp1 (1:100; Abcam, Cambridge, UK), anti-Gαi3 (1:200; Sigma, St Louis, MO, USA), anti-Par6b (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ZO-1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-spectrin (1:200; BD Biosciences, San Jose, CA, USA), anti-β-catenin (1:100; Cell Signaling, Danvars, MA, USA), anti-α-tubulin (1:200; Sigma, St Louis, MO, USA), anti-Cdh23 (1:200; Abcam, Cambridge, UK) and anti-Vangl2 (1:200; R&D Systems, Minneapolis, UK). An LSM510 laser confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to observe the specimens at 63× magnification with excitation wavelengths of 647, 555 and 488 nm. The scanning aperture was 1 unit, the linear average was 4 times, the scanning speed was 7 and the image resolution was 1024 × 1024. Confocal microscopy images were z-stacks of all planes in which the proteins expressed and were processed with Adobe Photoshop.