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. 2013 Jul 31;110(33):13516–13521. doi: 10.1073/pnas.1311784110

Fig. 2.

Fig. 2.

PRMT5 binds to p65 and is responsible for NF-κB activation and dimethylation of R30. (A and B) Endogenous p65 coimmunoprecipitates with PRMT5. 293IL1R cells were treated with 10 ng/mL of IL-1β for 1 h or were untreated. PRMT5 binds to p65 only when NF-κB is activated. Reverse immunoprecipitation, using anti-PRMT5, gave a similar result. (C, Upper) Western assay showing either overexpression or shRNA-mediated knockdown of PRMT5 in 293IL1R cells. (Lower) Luciferase assay of NF-κB in 293IL1R cells treated with 10 ng/mL of IL-1β for different times, showing that reduced PRMT5 expression decreased and overexpression increased NF-κB activation. The data represent the means ± SD for three experiments. *P < 0.01 vs. Ctrl group. (D, Upper) Western assays, showing either overexpression (Upper Left) or shRNA-mediated knock down of PRMT5 (Upper Right) in colon cancer HT29 cells. (Lower) Luciferase assay of NF-κB in HT29 cells, showing that overexpression of PRMT5 further increased NF-κB activation, whereas reduced PRMT5 expression decreased NF-κB activation. The data represent the means ± SD for three experiments. *P < 0.01 vs. Ctrl group. (E) EMSA, showing that in response to IL-1β treatment, NF-κB DNA binding ability was significantly decreased in 293IL1R cells in which PRMT5 has been knocked down, compared with control cells (Left), whereas increased PRMT5 expression greatly enhanced NF-κB DNA binding ability (Right). (F) Coimmunoprecipitation and Western assays, showing that R30 of p65 is dimethylated by PRMT5 upon IL-1β treatment. Overexpression of PRMT5 increased the dimethylation of R30, but shRNA-mediated knock down had the opposite effect. (G) Luciferase assay of NF-κB activity, showing that the R30A mutation decreased NF-κB activation. Overexpression of PRMT5 activated NF-κB in control cells and in 293IL1R cells overexpressing WTp65, but not in these cells overexpressing the R30A mutant protein. The data represent the means ± SD from three experiments. *P < 0.01 vs. Ctrl group; †P < 0.01 vs. WTp65 group; #P < 0.01 vs. Ctrl, PRMT5 group; $P < 0.01 vs. WTp65, PRMT5 group.