NF-κB p65 detection in subcellular compartments
RAW264.7 cell was seeded in 6-well plates (1×106 per
well) and cultured with DMEM, with 10% fetal bovine serum. After 24 h,
Culture media was then renewed and cells were treated with or without
As2O3 (2.5 μM or 5.0 μM ) for 2-6 h or
BAY11-7082(5mM, MCE,
Cat#HY-13453) for 40 min, followed by LPS (200 ng
mL-1, Sigma, Cat#L2880) stimulation for another 30
min. The treated cells were washed with PBS and suspended in lysis
buffer A (10 mmol L-1 HEPES, pH 7.6; 10 mmol
L-1 KCl, 1 mmol L-1 dithiothreitol,
0.1 mmol L-1 EDTA and 0.5 mmol L-1PMSF) for 10 min on ice. Cytosolic fractions were separated using
pipettes after centrifugation at 12 000×g for 10 min at 4 °C. The
remaining unclear fractions were lysed again with lysis buffer B (20
mmol L-1 HEPES, pH 7.6; 1 mmol L-1EDTA, 1 mmol L-1 , DTT, 0.5 mmol L-1PMSF, 25% glycerol and 0.4 mol L-1 NaCl) and
centrifuged at 12 000×g for 20 min. As mentioned above,
anti-NF-κB p65
(1:1000 dilution, Biolegend,
Cat#622602,RRID:AB_315956) primary antibody was used to detect the
cytosolic and nuclear proteins by western blotting. GAPDH and PCNA were
used as internal controls for the cytoplasmic and nuclear fractions.