Anti β actin clone ac 15 a5441

Brain tissue was homogenized in an equal ratio of tissue homogenate buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 320 mM sucrose, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and lysis buffer (1% Triton, 10% glycerol, 120 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.75 mM MgCl₂, 2 mM NaF, 1 mM sodium vanadate, protease inhibitor cocktail). Alternatively, transfected HEK293T were washed in ice-cold PBS and lysed in Triton lysis buffer (1% Triton, 10% glycerol, 120 mM NaCl, 25 mM HEPES, 1 mM EDTA, 0.75 mM MgCl₂, 2 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors). Samples were incubated on ice for 10 min and then centrifuged at 11,000× g for 10 min at 4 °C to separate nuclei from residual tissue. Total protein concentration was determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA), and equal amounts were loaded on SDS-PAGE, transferred to a nitrocellulose membrane, and blocked in an Odyssey blocking buffer. Membranes were incubated with primary and secondary antibodies and imaged using the Odyssey CLx imaging system (LI-COR Biosciences, Lincoln, NE, USA). Anti-FAK (clone 77; 610088) was obtained from BD Biosciences. Anti-FLAG (clone M2; F3165) and anti-β actin (clone AC-15; A5441) were obtained from Sigma-Aldrich. Secondary antibodies (goat anti mouse 680LT and goat anti rabbit 800CW) were obtained from Li-COR Biosciences.