Protein Analysis

Ureters were dissected from E17.5 and E18.5 embryos and immediately frozen. Proteins were extracted using standard RIPA lysis buffer with phosphatase and protease inhibitors. Protein quantification was performed using the Bradford Assay and 1.0 μg/μl of protein lysate was placed onto each chamber of the PathScan Intracellular signaling array (Cell Signaling Technology #7744). Assay conditions and procedures followed the manufacturer’s recommendations. Florescence readout was performed using the Licor Odyssey Imager. PathScan data analysis was quantified using ImageStudio software provided by Licor Biosciences. The relative fluorescence unit (RFU) of each antibody spot was quantified according to the Cell Signaling protocol and normalized to the positive control antibody spots (also provided on the Cell Signaling array). Student t-test comparisons were performed using Prism GraphPad software.