Mouse monoclonal anti rhoa antibody

The corpus callosa were dissected under a Leica stereo microscope (MZ 6; Leica Biosystems, Buffalo Grove, IL), followed by washes in 1x PBS and lysis in ice-cold RIPA buffer (1% Nonidet P-40, 50 mM Tris pH 7.6, 120 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail set 1 (Millipore, Burlington, MA). The lysates were cleared of insoluble materials by centrifugation at 16,000 x g for 10 min at 4°C. Protein concentration was determined by a Bio-Rad protein assay method (Bio-Rad, Hercules, CA) according to the manufacturer's protocol, and equal amounts of protein were used for SDS–PAGE and western blot analysis. The GTP-Rho pull-down assay was performed as previously described (Luo et al., 2011 (link)); in short, tissues were pulverized on liquid nitrogen, lysed in 300 µl of ice-cold RIPA buffer containing protease inhibitors with a cell disruptor for 10 min and homogenized with a 26 G syringe needle. Equal amounts of total protein were incubated with 60 μg GST-RBD beads (Cytoskeleton, Denver, CO) at 4°C for 90 min. The beads were washed twice with lysis buffer and once with TBS buffer. Bound Rho proteins were eluted by Laemmli sample buffer and detected by western blot using mouse monoclonal anti-RhoA antibody (Cytoskeleton, Denver, CO).