Anti phospho erk1 2 t202 y204 antibody

Cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail) for 30 min on ice. Lysates were centrifuged at 2,0000 g for 30 min at 4 °C. The supernatant was mixed with SDS loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, and 20% glycerol with bromophenol blue) and heated for 5 min. Proteins were separated by 12% SDS-PAGE gel and transferred to PVDF membranes. The membrane was blocked in 5% non-fat milk, and incubated with the intended primary antibody in TBS containing 0.1% Tween 20 (TBS-T) for 3 h. After washing with TBST-T, the membrane was incubated with HRP-conjugated secondary antibody for 1 h. After three washes with TBST-T, bands were visualized by chemiluminescence. The primary antibodies used in this study were: as follow anti-transgelin-2 (Novus Biologicals, CO, USA), anti-β-actin (Cell Signaling Technology, MA, USA), anti-Flag (Sigma-Aldrich, MO, USA), anti-GFP (Abcam, MA, USA), anti-ERK1/2 (Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2-T202/y204 antibody(Cell Signaling Technology, MA, USA), anti-β-tubulin antibody (Abcam, MA, USA), anti-GST antibody (Abcam, MA, USA).

Total protein and nuclear fraction were extracted from cardiomyocytes and heart tissue and Western blotting was performed as previously described [52 (link),55 (link)]. Ten µg of each fraction were resolved by SDS-PAGE. For Western blotting, anti-phospho-ERK1/2 (T202/Y204) antibody (Cell Signaling Technology, Danvers, MA, USA), anti-ERK1/2 antibody (Cell Signaling Technology), anti-phospho-GATA4 (S105) antibody (Abcam, Cambridge, United Kingdom), and anti-GATA4 antibody (Cell Signaling Technology) were used as primary antibodies, and anti-mouse antibody (MBL, Aichi, Japan) and anti-rabbit antibody (MBL) were used as secondary antibodies. Western blotting signals were visualized with an Amersham Imager 680 blot imager (GE Healthcare, Chicago, IL, USA) and quantified with NHI ImageJ software (version 1.52a).