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. 2012 Jun 14;287(35):29213–29226. doi: 10.1074/jbc.M112.355545

FIGURE 4.

FIGURE 4.

JNKBP1 attenuates NOD2-mediated NF-κB activation. A, JNKBP1 in a dose-dependent manner inhibits NOD2-mediated NF-κB activation. HEK293T cells were transfected with the reporter plasmid (κB)5-LUC and increasing amounts of JNKBP1-V5 along with (black bars) or without (white bars) NOD2. 24 h post-transfection, cells were harvested for LUC assays. Values represent the means ± S.D. (error bars) of triplicate cultures. Immunoblot with an anti-V5 antibody shows the JNKBP1-V5 expression level. B, effect of JNKBP1 on TNFα- and MDP- induced NF-κB activation. HEK293T cells were transfected with the reporter plasmid (κB)5-LUC and increasing amounts of JNKBP1-V5 in the presence (white and black bars) or absence (gray bars) of NOD2. 24 h post-transfection, cells were left untreated (white bars) or were stimulated for 16 h with MDP (1 μg/ml) (black bars) or with TNFα (100 units/ml) (gray bars) before being harvested for LUC assays. Values represent the means ± S.D. of triplicate cultures. Immunoblot with an anti-V5 antibody shows the JNKBP1-V5 expression level. C, efficiency of specific siRNA 4 to target JNKBP1 expression. HEK293T cells were transfected with JNKBP1-V5 along with either the siRNA 4 JNKBP1 or a siRNA control. After 48 h, cell lysates were analyzed by immunoblotting with an anti-V5 antibody. Equal loading was monitored by concomitant detection of β-tubulin. D, knockdown of JNKBP1 increases MDP-induced NF-κB activation. GNV cells were transfected with the siRNA 4 JNKBP1 or the siRNA control. After 24 h, the cells were transfected with the reporter plasmid (κB)5-LUC, and following another period of 24 h, cells were treated with MDP (1 μg/ml) for 16 h before being harvested for LUC assays (mean ± S.D. (n = 3); *, p < 0.04). E, knockdown of JNKBP1 up-regulates MDP-induced IKKα/β, IκBα, and p65 phosphorylation. HCT116 cells were transfected with JNKBP1 siRNA 4 or 6 or the siRNA control. After 48 h, cells were treated with MDP (60 μg/ml) for the indicated periods. The lysates were analyzed by immunoblotting with the following antibodies: anti-NOD2, anti-JNKBP1, anti-phospho-Ser176/177 IKKα/β, anti-phospho-Ser32/36 IκBα, and anti-phospho-Ser536 p65. Equal loading was monitored by concomitant detection of β-tubulin. RLU, relative light units.