Ab135694

Samples of feline cerebral cortex (5 -10 mg) were homogenized in 400 µl of RIPA buffer (PIERCE, ThermoFisher, Waltham, MA, USA) with a hand-held micro-pestle for 30 sec, followed by passage through a 25G needle and kept on ice for 20 min. After centrifugation at 16,200 ×g for 30 min at 4˚C, the soluble fraction was transferred to a new tube and total protein concentration was determined by DC protein assay (Bio-Rad, Hercules, CA, USA). Following quantification, 33 µg of protein sample were mixed with 4× Laemmli sample buffer (Bio-Rad) containing 400mM Dithiothreitol. Protein fractions were separated with 18 % sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to Odyssey® nitrocellulose membrane (Li-Cor, Lincoln, NE, USA), blocked in LI-COR Odyssey blocking buffer (Lincoln, NE) for 1 h, and incubated with rabbit polyclonal anti-PEA-15 (C-terminal amino acids 93-123 of Human PEA15) antibody ab135694 (Abcam, Cambridge, UK) at a concentration of 1:100 and anti-GAPDH antibody (MAB374, EMD Millipore, Burlington, MA, USA) at a concentration of 1:500. Secondary antibodies (1:15,000) were IRDye®680RD Goat anti-Rabbit IgG (H+L, Li-Cor) and IRDye®800CW Goat anti-Mouse IgG (H+L, Li-Cor), respectively. The fluorescent signal was detected using Odyssey® Infrared imaging system (Li-Cor).