Rabbit anti erk1 2 monoclonal antibody

Cells were lysed in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 12% glycerol, 2% SDS, 0.004% bromophenol blue and 5% 2-mercaptoethanol). The samples were separated by SDS-PAGE on SuperSep Ace 5-20% or SuperSep Ace 5-20% pre-cast gels (Wako Pure Chemical), and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). After 60-min incubation with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) at room temperature, the membranes were incubated for overnight at 4°C with rabbit monoclonal anti-ERK1/2 antibody (1:1,000) (No. 9101; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-ROCK1 antibody (1:1,000) (ab45171; Abcam, Cambridge, MA), rabbit monoclonal anti-ROCK2 antibody (1:1,000) (ab125025; Abcam), mouse Anti-ERK antibody (1:1,000) (No. 610123; BD biosciences, San Jose, CA) or mouse monoclonal anti-GFP antibody (1:8,000)(No. 632381, Clontech Laboratories, Inc., Mountain View, CA) . The immunoreactivities were visualized with IRDye 800CW-conjugated donkey antimouse IgG antibodies (1:15,000; LI-COR) and IRDye 680LT-conjugated goat anti-rabbit IgG antibodies (1:15,000; LI-COR). All antibodies were diluted in Odyssey blocking buffer. Proteins were detected by an Odyssey Infrared Imaging System (LI-COR) and analyzed by using the Odyssey imaging software.