p300 and CBP protect VRK1 from p53-induced downregulation.

<p>(A) H1299 cells were transfected with pCB6+p53 (0.2 µg) and pCEFL-HA-VRK1 (5 µg) with increasing amounts of pCMVβ-p300-HA and 36 hours postransfection cell extracts were analyzed with the different antibodies indicated in the legend. Normalized VRK1 protein levels are represented in the graphs. VRK1 and p300 were detected with an anti-HA antibody. (B) p300 over-expression by itself does not have any effect on VRK1 protein levels. An experiment similar to that in part A, but in the absence of p53 was performed. (C) H1299 cells were transfected with pCB6+p53 and pCEFL-HA-VRK1 with increasing amounts of pSG5-CBP and 36 hours postransfection cell extracts were analyzed with the different antibodies indicated in the legend. CBP was detected with a rabbit polyclonal antibody. Normalized VRK1 protein levels are represented in the graphs. (D) CBP over-expression by itself does not have any effect on VRK1 protein levels. An experiment similar to that in part C, but in the absence of p53 was performed. Each protein was detected in the immunoblots as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002649#s4" target="_blank">methods</a> section. (E). Negative control for lack of p53 effect. H1299 cells were transfected with pCEFL-HA-TSG101 (5 µg) and increasing amounts of pCB6+p53 (0.2 µg). TSG101 was detected with an anti-HA antibody. The experiments were performed independently three times. The quantifications corresponding to immunoblots with differences (parts A–D) are shown and presented in bar graphs.