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. 2010 Dec 7;286(7):5727–5735. doi: 10.1074/jbc.M110.108001

FIGURE 5.

FIGURE 5.

DFK1012 does not block NF-κB subunit p65 nuclear translocation upon LPS or CpG-B stimulation. A, RAW 264.7 cells or BMDM were pretreated with DFK1012 (DK12, 30 nm) for 1 h and then stimulated with 1 ng/ml of LPS for 30 min. Cells were fixed and permeabilized for 5 min. Immunofluorescent staining for p65 was performed using anti-p65 antibody followed by Alexa Fluor 594 detection antibody. Cells were analyzed using fluorescence microscope. B, BMDM were pretreated with DFK1012 (30 nm) for 1 h and then stimulated with CpG-B (1 μm) for 30 min. Equal amounts of nuclear and cytoplasmic protein extracts were subjected to Western blot analysis for p65. CE, cytoplasmic extracts; NE, nuclear extracts. C, RAW 264.7 cells were pretreated with DFK1012 (30 nm) for 1 h and then stimulated with LPS (10 ng/ml) for 30 min or treated with DFK845 (DK5) for 90 min. Cells were incubated in Hepes-Triton buffer (pH 7.4) to isolate pure nuclei and analyzed for p65/NF-κB localization by flow cytometry after staining with FITC-labeled anti-p65 antibody. Data are representative of three independent experiments with similar results. ctrl, control.