Figure 3: Failure to terminate TNF-induced phosphorylation of p65 by PHF20.

(a,b) HeLa and D54 cells were infected with either rAd–Lac or rAd–flag–PHF20 for 24 h, and then cells were treated with TNF for indicated times. Whole-cell lysates were immunoblotted with indicated antibodies (left). Relative density was obtained by densitometry of the corresponding immunoblot data. Data are from one representative of three independent experiments (mean±s.e. of triplicate, (right). (c) Teb-on PHF20 HEK293 cells were incubated with or without teb (25 μM) for 12 h, and then further treated with TNF for indicated times. Whole-cell lysates were immunoblotted as in a. (d) HEK293 cells were co-transfected with the expression plasmids of HA-p65, Myc-p300 and different amounts of flag–PHF20. Immunoprecipitates were prepared with anti-HA antibody and immunoblotted with anti-Acetyl-p65-Lys310 antibodies. Whole-cell lysates were immunoblotted as in a. (e) HeLa cells were infected with either rAd–Lac or rAd–flag–PHF20, which was followed by TNF treatment for indicated times. After the immunoprecipitation with anti-p65 antibody, immunoprecipitated IκBα was detected by immunoblotting with anti-IκBα antibody (top first row). Co-immunoprecipitated p50 serves as a positive control (top second row). Whole-cell lysates were immunoblotted as in a. The asterisk indicates nonspecific bands for anti-IκBα antibody. (f) HEK293 cells were co-transfected with the indicated expression plasmids, and followed by TNF treatment for 2 h. Immunoprecipitates were prepared with anti-p65 antibody and immunoblotted as in e. (g) HeLa cells were infected with either rAd–Lac or rAd–flag–PHF20, and followed by TNF treatment for indicated times. Cytosolic and nuclear fractions were obtained, and immunoblot was performed as in a.