Western blotting analyses of NF-κB and MAPK signaling pathways in BMDMs after LPS stimulation.
(A) PLT-BMDMs and control BMDMs (2.5 × 106 cells) were stimulated with LPS (50 ng/mL) for 0–120 min. Cells were then lysed in 1 × SDS sample buffer, and the cell lysates were subjected to western blotting analysis with antibodies against phospho-IκBα, total IκBα, phospho-NF-κB p65, total NF-κB p65, phospho-p38 MAPK, total p38 MAPK, phospho-JNK, total JNK, phospho-ERK1/2, total ERK1/2 or GAPDH. The relative intensity of each band after normalization to the corresponding level of GAPDH is shown in the right panel. Experiments were repeated three times, and representative results are shown. (B) PLT-BMDMs and control BMDMs (2.5 × 106 cells) that had been stimulated with LPS (50 ng/mL) for 0–120 min were separated into cytoplasmic and nuclear fractions. Each fraction was subjected to western blotting analysis with antibodies against NF-κB p65. GAPDH and histone H3 were used as controls for the cytoplasmic and nuclear fractions, respectively. The relative intensity of each band after normalization to the level of GAPDH or histone H3 is shown in the lower panel. Experiments were repeated three times, and representative results are shown.
