Alexa fluor 680 goat anti mouse

Some of the activated kinases revealed by PamChip array were validated by western blot analysis as described (with modifications; Fuhler et al., 2009; Queiroz et al., 2012). Briefly, cell lysates (40 µg) were prepared as for kinome profile analysis, mixed with 2× Laemmli buffer, separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane (Immobilon FL membrane; Merck KGaA, Darmstadt, Germany) and nonspecifically blocked with Odyssey buffer (LI‐COR Biosciences, Lincoln, NE). Membranes were incubated overnight with primary antibodies against pFAK (Y925; rabbit polyclonal; Signalway Antibody, College Park, MD), pERK, pPKB, pEGFR, and pSMAD1/5/8 (Cell Signaling Technology, Beverly, MA) and β‐actin loading control (mouse monoclonal; Clone sc‐47778; Santa Cruz Biotechnology, Dallas, TX). All antibodies were used 1:1,000. Membranes were probed with secondary antibody conjugated with goat‐anti‐mouse‐Alexa Fluor 680 and goat‐anti‐rabbit IRDye 800CW at 1:5,000 (LI‐COR Biosciences). Odyssey infra‐red imaging (LI‐COR Biosciences) was used to detect proteins. Quantification was performed using Odyssey 3.0 Software, LI‐COR, Lincoln, NE.

Following treatments, cells were fixed with 4% NBF for 15 minutes before incubation with blocking buffer (phosphate-buffered saline, normal goat serum, water, and Triton X-100). Cells were treated with rabbit polyclonal anti-CXCL4 (1:25; Abcam) and mouse monoclonal anti–β-tubulin (1:1000; Sigma-Aldrich) antibodies overnight at 4°C. Cells were washed before incubation with goat anti-rabbit IRDye 800CW (Molecular Probes, Eugene, OR) and goat anti-mouse Alexa Fluor 680 (LI-COR Biosciences, Lincoln, NE). The LI-COR Odyssey IR Imaging System was used to analyze results.