|Background||Cell line: A549 (Human lung carcinoma)|
Growth media: DMEM and 10% NCS (Newborn calf serum)
A549 cell lysate was prepared by homogenization in modified RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.4, 1 mM ethylene diamine tetraacetic acid, 1 mM phenyl methyl sulfonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecyl sulfate, 5 μg/ml of aprotinin, 5 μg/ml of leupeptin). Cell debris was removed by centrifugation. Protein concentration was determined with Bio-Rad protein assay. The cell lysate was boiled for 5 min in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol.