Protran premium nitrocellulose membrane

Cells were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail [Roche]). The amount of protein was quantitated using a BCA kit (Thermo) [7 (link)]. Then, equal amounts of protein were resolved on 10% Tris-glycine SDS-PAGE and transferred onto Protran^® Premium nitrocellulose membrane (GE healthcare). The membrane was then probed with mouse anti-cortactin (1:500; #05-180, clone 4F11, Millipore), rabbit anti-cortactin (1:500; #3502, Cell Signaling), rabbit anti-GFP (1:1000; #632592, Takara), rabbit anti-β-actin (1:2000; #ab8227, Abcam) or goat anti-NP (1:10,000; G150, kindly provided by Richard Webby, St Jude Children’s Research Hospital, Memphis (TN), USA) antibody followed by horseradish peroxidase-conjugated anti-mouse IgG (1:5000; #626520, Life Technologies), anti-rabbit IgG (1:5000; #A16023, Life Technologies) or IRDye 680RD-conjugated anti-goat IgG (1:50,000; #926-32214, LI-COR) antibody. The protein bands that were visualized by chemiluminescence or fluorescence and images were acquired on Odyssey Fc imaging system (LI-COR). Images were then exported as TIFF files, minimally adjusted for brightness and contrast, and composed in figures in Adobe Photoshop CC 2015 [7 (link),15 (link)].

Protein samples were separated by SDS-PAGE electrophoresis (1.0 mm, 4%–12% gradient, Novex NuPAGE Bis-Tris Gel; Life Technologies) and transferred onto a Protran Premium nitrocellulose membrane (GE Healthcare). The primary antibodies used in this study are listed in Table S1. After incubation with corresponding secondary IRDye-800CW-conjugated antibodies (1:10,000; LI-COR Biosciences), infrared fluorescence was read on an Odyssey Imaging System (LI-COR Biosciences). Band intensities were measured using Odyssey application software (Image Studio Lite, version 4.0; LI-COR Biosciences).