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. 2010 May 27;38(18):6029–6044. doi: 10.1093/nar/gkq439

Figure 8.

Figure 8.

TNF-induced recruitment of IKKε to PML-NBs. (A) HeLa cells were transfected to express the PML splicing forms PML-III, PML-IV or the PML-RARα fusion protein. Thirty-six hours after the transfection, cells were left untreated or stimulated with TNF for 30 min. The subcellular localization of PML and endogenous IKKε was visualized by indirect immunofluorescence using an anti-PML (red) and anti-IKKε antibody (green). (B) Upper: Hela cells transfected with a plasmid expressing a PML-specific shRNA were treated with TNF for 30 min as shown and analyzed for subcellular localization of endogenous IKKε using an anti-IKKε antibody. Lower: the efficient knock-down of PML was controlled by transfection of the vector directing the synthesis of the PML specific shRNA and detection of PML by immunofluorescence. The limited transfection efficiency allows to observe PML down-regulation only in transfected cells. (C) Cells were transfected with a vector for a PML specific shRNA or a control. After 36 h, cells were stimulated for 30 min with TNF as shown and the localization of the endogenous proteins was revealed by immunofluorescence. (D) Hela cells were treated as in (C) and analyzed for the occurrence and subcellular localization of endogenous PML and serine 468 phosphorylated p65 as shown. (E) Schematic model summarizing the mechanisms and functions of p65 phosphorylation and its phosphorylation by IKKε and other kinases.