Figure 5. Overexpression of ProT increases NF-κB acetylation.

(a) Detection of acetylated NF-κB p65 (Lys310). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviruses expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression vector (pCMVb p300 HA delta33) or a control vector (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression in the transfected cells were verified. (b) Reporter assay for NF-κB transactivation activity. 293T cells that had been transduced with lentiviruses expressing ProT-IRES-GFP or GFP alone (left), or ProT or GFP shRNA (right) were cotransfected with pNF-κB-Luc and pTK-Renilla reporter plasmids. Total cell lysates were harvested 48 h later, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light activity. Values shown are the mean±s.e.m. (n=4 for left and n=5 for right; Student’s t-test). (c) NF-κB and ProT binding assay. Cell lysates from 293T cells transduced with lentiviruses expressing GFP-tagged ProT or GFP alone were immuoprecipitated by anti-GFP monoclonal antibody followed by immunoblotting with anti-NF-κB p65 and anti-GFP antibody. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of NF-κB and ProT by immunoblotting. (d) Analysis of the effect of ProT on NF-κB and HDAC3 binding. 293T cells that had been transfected with an NF-κB p65 expression vector (pcDNA3-cFlag-RelA) or a control vector (pCMV-Tag2B) (left) or HDAC3 expression vector (pcDNA3.1-HDAC3-Flag) or a control vector (pCMV-Tag2B) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. After 24 h, total cell lysates were immuoprecipitated by Flag-M2 beads followed by immunoblotting with the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated by immunoblotting with indicated proteins. (e) p300 and NF-κB binding assay. 293T cells that had been transduced with lentiviruses expressing ProT shRNA-2 or luciferase shRNA as the control were transfected with the pCMVb p300 HA delta33 and pcDNA3-cFlag-RelA. Total cell lysates were immunoprecipitated by anti-HA antibody followed by immunoblotting with the indicated proteins. Results are representative of three independent experiments. (f) Proposed model for ProT-mediated NF-κB hyperacetylation via HDAC3/NF-κB dissociation and p300/NF-κB association.