Versa doc model 1000

Astrocytes were pelleted and resuspended in lysis buffer (pH 7.5) containing: (in mM) Tris-HCl 20, NaCl 150, EDTA 1.0, EGTA 1.0, PMSF 1.0, 1% Triton X-100, and an additional mixture of peptide inhibitors (leupeptin, bestatin, pepstatin, and aprotinin). Total protein of homogenates were determined with the Bradford protein assay (Bio-Rad), followed by addition of an appropriate volume of Urea sample buffer (62mM Tris/HCl pH 6.8, 4% SDS, 8M Urea, 20mM EDTA, 5% β-Mercaptoethanol, 0.015% Bromophenol Blue) to load 15 μg of protein per lane. Next, samples were boiled in a water bath at 95 °C for 10 minutes, spun to pellet debris, and immediately run on 7% SDS-polyacrylamide gels. Western blotting was performed as previously described (Kucheryavykh et al., 2009 ) using rabbit polyclonal antibodies against AKAP150 (1:200; Santa Cruz Biotechnology, Dallas, TX, USA; Cat# sc-10765, RRID:AB_2289482). Final detection was performed with enhanced chemiluminescence methodology (SuperSignal® West Dura Extended Duration Substrate; Pierce, Rockford, IL, USA) as described by the manufacturer, and the intensity of the signal was measured in a gel documentation system (Versa Doc Model 1000, Bio Rad, Hercules, CA, USA). In all cases, intensity of the chemiluminescence signal was corrected for minor differences in protein content after densitometry analysis of the India ink stained membrane.