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. 2015 Aug 8;6(30):29396–29412. doi: 10.18632/oncotarget.5120

Figure 2. YBX1 is an activator of NF-κB and phosphorylation of S165 is critical for its NF-κB activating ability.

Figure 2

Figure 2

A. Western Blot, left panel, showing that YBX1 is knocked down by shRNAs. Right panel, showing that both wild type (WT-) YBX1 and mutant S165A-YBX1 were overexpressed at similar levels in 293 cells. B. κB-specific luciferase assay in 293 cells, top panel, NF-κB luciferase assay, showing that knockdown of YBX1 decreased IL-1β-induced NF-κB activation. The data represent the means ± SD from three independent experiments. *P < 0.05 vs. Ctrl group; #P < 0.05 vs. Ctrl+IL-1β group. Bottom panel inset, showing that overexpression of WT-YBX1 could activate NF-κB, while overexpression of S165A-YBX1 dramatically reduced NF-κB activation. Bottom panel main figure, showing that overexpression of WT-YBX1 could further enhance IL-1β-induced NF-κB activation, while overexpression of mutant S165A-YBX1 led to significantly lower NF-κB activation as compared to that of WT-YBX1. The data represent the means ± SD from three independent experiments. * P < 0.05 vs. Ctrl group; # P < 0.05 vs. WT-YBX1 group. C. κB-specific luciferase assay, showing that either WT-P65 or WT-YBX1 could activate NF-κB respectively. Additionally, WT-P65 and WT-YBX1 have synergistic effect on the activation of NF-κB. Mutant S165A-YBX1 showed much compromised ability for this synergistic effect. The data represent the means ± SD from three independent experiments. *P < 0.05 vs. Ctrl group. #P < 0.05 vs. WT-P65 group. $P < 0.05 vs. WT-P65/WT-YBX1 group. D. Western Blot in colon cancer HT29 cells, top left panel, showing that YBX1 was successfully knocked down by a pool of YBX1 specific shRNAs. Top right panel, showing that both WT-YBX1 and mutant S165A-YBX1 were overexpressed at similar levels in HT29 cells. Anti-Flag antibody was also used to show that both Flag-tagged WT-YBX1 and S165A-YBX1 was expressed at similar levels. Bottom left panel, showing that using shRNA to knock down YBX1 decreased NF-κB activity as compared to the HT29 control cells. The data represent the means ± SD for three independent experiments. *P < 0.01 vs. Ctrl group. Bottom right panel, κB-specific luciferase assay, showing that overexpression of mutant S165A-YBX1 led to decreased NF-κB activity as compared to that of WT-YBX1 cells. The data represent the means ± SD for three independent experiments. *P < 0.01 vs. Ctrl group; #P < 0.01 vs. WT-YBX1 group.