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. 2015 Sep 28;5:14470. doi: 10.1038/srep14470

Figure 3. Activation of NF-κB via ATF3 down-regulation.

Figure 3

(a) Cells were transfected with 100 nM ATF3 small interfering RNA (siRNA) and reagent alone for 48 h and then treated with 1 μg/mL LPS for the indicated times. The protein levels of ATF3, p65, α-tubulin, and topoisomerase II-α were detected with Western blot using the respective antibodies. α-tubulin and topoisomerase II-α were used as a loading control for cytoplasm and nucleus, respectively. (b) p65 and ATF3 were assessed with confocal microscopy. Cells were transfected with 100 nM ATF3 siRNA and reagent for 48 h and then treated with 1 μg/mL LPS for 2 h. (c) RAW 264.7 cells (left panel) were transfected with ATF3 siRNA and reseeded from a 10-cm dish to a 96-well plate to quantify NF-κB activity using enzyme-linked immunosorbent assay (ELISA). Human embryonic kidney 293 (HEK293) cells (right panel) were also transfected with ATF3 siRNA, and NF-κB activity was detected with SEAP assay using ELISA. Data represent three independent experiments (n = 3; *P < 0.05, **P < 0.01). (d) Cells were treated with 1 μg/mL LPS for the indicated times. Beads were bound with p65 antibody, and p65 and HDAC1 were precipitated. A total of 20 μg protein was separated with electrophoresis. Proteins levels were determined with Western blot using whole-cell lysates. (e) Cell lysates were processed with NE-PER lysate, and the protein expression levels of ATF3, ac-p65, HDAC1 and β-actin were measured by Western blot. β-actin and HDAC1 were used as a loading control for cytoplasm and nucleus, respectively. (f,g) Human PBMCs were transfected with 100 nM ATF3 siRNA and reagent alone for 48 h and then treated with 1 μg/mL LPS for the indicated times. (f) The protein levels of ATF3 and β-actin were detected by Western blot; β-actin was used as a loading control. (g) The protein expression levels of ATF3, ac-p65, topoisomerase II-α, and β-actin were measured with Western blot. β-actin and topoisomerase II-α were used as a loading control for cytoplasm and nucleus, respectively. Western data show a representative set among the results of three independent experiments (n = 3).