Irdye 700 nf κb consensus oligonucleotide

Nuclear fractionation was first performed on cells to be assayed. Cells were washed twice with ice-cold PBS and the lysed in 1 ml lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF). Lysates were incubated for 15 min on ice. 62.5 μl 10% NP-40 was added and samples were placed on a shaker (2 min at 4°C). Samples were centrifuged (1 min; room temperature; 13,000 rpm) and the nuclei pellet was re-suspended in 100 μl high salt buffer (20 mM HEPES pH 7.9, 4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) followed by rough shaking for 20 min at 4°C, and centrifugation (5 min; 4°C; 13,000 rpm). Supernatants were transferred to a new tube and protein concentration was determined. IRDye 700 NF-κB Consensus Oligonucleotide and Odyssey Infrared EMSA Kit were purchased from LI- COR Biosciences. For mobility shift assays, 5 μg of nuclear protein was incubated on ice in 20 μl of a buffer containing 10 mM HEPES (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiotreitol, 0.1% NP-40 and 0.05 mg/ml poly (dI-dC). Labeled probe was added 30 min before gel loading. Samples were resolved on a non-denaturing 5% polyacrylamide gel in 0.5x TBE. Imaging was performed on the Odyssey (LI-COR Biosciences) using the 700 nm channel.