|Sub-confluent cells were treated with 0, 2.5 and 5μM of OSU-T315 for 48 and 72 hours for cell lines, and 72 hours for primary cells. Cells were then harvested and homogenized in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris, pH 8.1) supplemented with protease/phosphatase inhibitor cocktail (Thermo Scientific, Waltham MA).
|Electrophoresis was performed with 25μg or 50μg of protein/lane in a 4-20% gradient SDS- polyacrylamide gel (Thermo Scientific, Waltham MA). Proteins were transferred to PVDF membranes (Millipore, Billerica MA), and probed with specific antibodies of interest and horseradish peroxidase-conjugated secondary antibodies.
|The chemiluminescent signals were detected in X-ray films.
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