Nuclear Protein Extraction and Immunoblotting

Harvest and pellet BMDM cells. Resuspend cell pellet in 250 µL ice cold lysis buffer (10 mM Hepes pH 7.9 + 10 mM KCl + 0.1 mM EDTA + 0.4% Nonidet P40) with protease inhibitors (Roche, 1836145) and incubate on ice for 15 min. Spin the lysate for 1 min at full speed at 4°C. Transfer supernatant to a fresh tube to keep the cytoplasmic fraction. Wash the nuclear pellet in 500 µL ice cold lysis buffer and spin down for 1 min at 4°C. Add 20 µL extraction buffer (20 mM Hepes pH 7.9 + 0.4 M NaCl + 1 mM EDTA) with inhibitors to the pellet and shake vigorously for 15 min on vortex in cold room. Spin for 10 min at full speed at 4°C. Transfer supernatant to fresh tube and store this nuclear extract at −20°C. Denature protein before use. Immunoblotting was performed as described previously (27 (link)). Anti-IκBα (9247), -phospho-IκBα (9246), -ERK (4372), -phospho-ERK (4695), and -NF-κB-p65 (8242) antibodies were obtained from Cell Signaling (Danvers, MA, USA). Anti-β-actin (ab8224) and -PCNA (ab152112) antibodies were from Abcam (Cambridge, MA, USA).