Western Blot Analysis of Cerebellum Proteins

Protein samples were isolated from cultured mouse mesothelial cells and from mouse tissue. Cells were grown in 25 cm² flasks and harvested at confluence. Cytosolic protein fractions were collected as described before [15 (link)]. Freshly excised mouse cerebellum was frozen in liquid nitrogen and homogenized in extraction buffer (10 mM Tris, 2 mM EDTA, 1 mM β-mercaptoethanol; pH 7.4) containing a cocktail of different protease inhibitors (Roche, Mannheim, Germany). Proteins (100 μg) from each cell culture sample, 1 μg protein from cerebellum, as well as 40 ng of purified human recombinant CR were loaded onto SDS-polyacrylamide gels (10 %). After separation, proteins were blotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) and incubated for two days at 4 °C with the CR-specific antibody CR7699/4 (Swant, Marly, Switzerland) at a dilution of 1:10,000. Rabbit secondary antibody linked to horseradish peroxidase (Sigma-Aldrich) was diluted at 1:10,000 and incubated for 2 days; prolonged incubation was shown to enhance the sensitivity in Western blotting [16 (link)]. For the detection, the chemiluminescent reagent Luminata Classico Forte (EMD Millipore Corporation, Billerica, MA, USA) was used. Chemiluminescent and normal illumination digital images were recorded on a system from Cell Biosciences (Santa Clara, CA, USA).