Research Paper Volume 18 pp 67—81

Effects of TGF-β1 and p38 MAPK inhibition on H4K16ac. (A) p-p38 MAPK levels in IMR90 24h after treatment with TGF-β1 (2ng/ml) with or without 2h pretreatment with SB202190, or with inhibitor alone. Whole cell lysates were prepared; β-actin was used as a loading control. (B) Nuclear extracts from cells prepared as in (A) were subjected to western blotting. H4 was used as a loading control. Numbers indicate densitometric ratio of p-p38 to total p38, or H4K16ac to H4. (C, D) ChIP assays with H4K16ac pulldown showing association with α-SMA (C), and Col3A1 (D) promoter regions. Quantitative ChIP assays were performed to analyze the association of H4K16ac with α-SMA or Col3A1 at the conditions indicated in A. DNA were crosslinked with the immunoprecipitated protein using specific antibody H4K16ac. Quantitative PCR was analyzed using 2^−ΔΔCt method, with results normalized to input DNA relative to vehicle control (Ctrl). Bar graphs represent mean ± SE from the average of at least three independent experiments; ^*p<0.05 compared to vehicle control.