Immunoblotting Workflow for Protein Analysis

Immunoblotting was performed as described^{2 (link)} with antibodies obtained from Cell Signaling Technologies: EphA1 (rabbit monoclonal D6V71), EphA2 (rabbit monoclonal D4A2), EphA3/A4/A5 (rabbit monoclonal D2C11), Erk1/2 (rabbit monoclonal 137F5), phospho-Erk1/2 (rabbit monoclonal 20G11), Stat1 (rabbit monoclonal 43H3), phospho-Stat1 (rabbit monoclonal D4A7), Stat3 (rabbit monoclonal D3Z2G), phospho-Stat3 (rabbit monoclonal D3A7) and cleaved Notch1 (rabbit monoclonal D3B8). Antibodies were used at a 1:1000 dilution of the supplied stock following the protocols specified by the supplier. Blots were developed using Thermo Fisher ECL Plus and visualized with a My ECL imager. Image acquisition time was adjusted to visualize all bands without saturating the brightest bands (from 30 seconds to 30 minutes depending on the efficacy of the antibody). Data were analyzed using Thermo My Image Analysis software, version 2.0. Images were inverted to display dark bands on a light background and in some cases brightness or contrast were adjusted to show lighter bands more distinctly, again without saturating the darkest bands. Images were then cropped, arranged and converted to TIFs with Photoshop, version CS6.