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. 2013 May 21;4:1906. doi: 10.1038/ncomms2906

Figure 3. Overexpression of ProT inhibits HDACs and enhances protein acetylation.

Figure 3

(a,b) Immunohistochemical staining for HDACs. Lung tissues from patients with emphysema of varying severity and non-COPD individuals (a), as well as lung tissues from 4-day-old ProT HZ, HET and NT mice (b) were immunostained with monoclonal antibody against HDAC1, HDAC2 and HDAC3. Note that lower levels of HDAC2, but not HDAC1 and HDAC3, were detected in patients with more severe emphysema and in ProT HZ mice, as compared with their normal counterparts. The boxed areas in upper panels (original magnification × 400; scale bar=100 μm) are magnified and shown in lower panels. (c) Immunoblot analysis of HDACs in the lung tissues of 4-day-old ProT HZ, HET and NT mice. (d) Detection of acetylated-lysine (acetyl-K). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviral vectors expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression plasmid (pCMVb p300 HA tag) or a control plasmid (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression were verified. (e) Detection of acetylated histones. 293 T cells were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 and their total cell lysates were examined for the indicated proteins by immunoblotting after 24 h. (f) HDAC and histone binding assay. 293T cells that had been transduced with HDAC1-Flag (pcDNA3.1-HDAC1-Flag) (left) or HDAC2-Flag (pCMV-Tag4C-HDAC2) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. Total cell lysates were immunoprecipitated by Flag-M2 beads and analysed by immunoblotting for the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of histone H1 and H3, HDAC1, HDAC2 and ProT by immunoblotting. (g) Proposed model for ProT-mediated enhancement of histone acetylation via HDAC/histone dissociation. Results are representative of three independent experiments.

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