Journal: PLoS ONE

Article Title: Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes

doi: 10.1371/journal.pone.0166710

Figure Lengend Snippet: A) Tyrosine phosphorylation of the high-MW Gab1 was analyzed by immunoprecipitation of the heart lysates. Mouse heart lysates were prepared at 5 min after injection with the cytokines and growth factors listed at top. Heart lysates were subjected to immunoprecipitation with anti-high-MW Gab1 serum, followed by western blot analysis using the antibodies indicated at the left. Arrowheads denote the high-MW Gab1. B) Activation level of ErbB4 was assessed by phospho-specific antibody. C) Activation levels of ERK1/2 and AKT were assessed by phospho-specific antibodies. Heart lysates were subjected to western blot analysis with the indicated antibodies. Representative blots of 3 experiments are shown. D) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 3). E) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 3). Values are shown as means±SEM for 3 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyze differences. * P <0.05, ** P <0.01, and *** P <0.001 for the indicated groups.

Article Snippet: Other antibodies were purchased as follows: anti-phospho-Gab1 (Tyr627) (1:1000, #3231), anti-Gab1 (1:1000, #3232), anti-phospho-AKT (Ser473) (1:1000, #9271), anti-AKT (1:1000, #9272), anti-phospho-p44/p42 (pERK1/2) (1:1000, #9101), anti-p44/p42 (ERK1/2) (1:1000, #9102), anti-pErbB4 (Tyr1284) (1:1000, #4757) antibodies for immunoblotting, and horseradish peroxidase-coupled horse anti-mouse (1:3000, #7076) and goat anti-rabbit IgG (1:3000, #7074) from Cell Signaling Technology; antibodies recognizing phospho-tyrosine (PY99) (1:1000, sc-7020), ErbB4 (1:400, sc-283), and SHP2 (1:400, sc-280) were from Santa Cruz Biotechnology Inc; anti-Gab1 (1:1000, 06–579) antibody for immunoblotting of immunoprecipitated proteins, and anti-p85 (1:1000, 06–195) antibody were from Millipore; antibodies against β-tubulin (mouse monoclonal) (1:3000, T5201), and anti-α-actinin (1:300, A7732) were from Sigma-Aldrich.

Techniques: Phospho-proteomics, Immunoprecipitation, Injection, Western Blot, Activation Assay