PMC:3537549 / 17837-19141 JSONTXT

{"project":"2_test","denotations":[{"id":"22906110-12414749-143530976","span":{"begin":522,"end":524},"obj":"12414749"}],"text":"Western blot\nCrFK and fcwf-4 grown to confluency in 12-well plates were stimulated with 600 μL of PBMC supernatants produced as explained above. At the time points indicated, cells were harvested and counted. 106 cells of either cell line were resuspended in 30 μL sample buffer (0.5 M Tris(hydroxymethyl)aminomethane, 5% SDS, 10% β-mercaptoethanol, 40% glycerol, and 0.05% bromphenol blue) and boiled at 95°C for 5 min. SDS-PAGE separation and submersed immunoblotting procedures were carried out as previously described[61]. The Spectra Multicolor Broad Range Protein Ladder (Fermentas GmbH, St. Leon-Rot, Germany) served as molecular weight standard marker for each blot. For protein visualization, membranes were first cut immediately below the 80kB marker band. The top and bottom membrane fractions were incubated with murine anti-human Mx MAb M143 (generously provided by Dr J. Pavlovic, Institute for Virology, University of Zürich, Switzerland) and murine anti-β-actin monoclonal antibody as a loading control (Sigma Aldrich GMbH, Buchs, Switzerland) respectively. Both fractions were subsequently incubated with a peroxidase-labelled goat anti-mouse IgG (Jackson Immunoresearch, Newmarket, Suffolk, UK). Bands were digitalized using the Chemigenius 2 BioImaging System (Syngene, Cambridge, UK)."}