Journal: Biochimica et biophysica acta
Article Title: Acetylation of lysine 109 modulates pregnane X receptor DNA binding and transcriptional activity
Figure Lengend Snippet: PXR is acetylated by p300 acetyltransferase. (A) Recombinant PXR was acetylated in vitro by incubating with either recombinant p300 or recombinant PCAF in acetylation buffer containing acetyl-CoA for 1 h at 30 °C. Acetylated PXR was detected by immunoblotting using an acetyl-lysine antibody and the blot was stripped and re-probed using a PXR antibody. (B) PXR was acetylated in vitro by p300 as described in (A), but this time in the presence of DMSO or 1 µM RIF and the reactions were quenched by boiling in Laemmli buffer at 0, 20, 40, or 60 min as indicated. (C) PXR protein was in vitro translated using the cell-free TnT coupled kit and subjected to in vitro acetylation assay using p300 as described in (A), with or without deacetylase inhibitors TSA and NAM. (D) 293T cells were co- transfected with FLAG-PXR and HA-p300 either separately or together. 24 h later, the cells were treated with DMSO or 25 µM RIF for an additional 24 h, nuclear extracts were harvested and subjected to FLAG-IP. To visualize FLAG-PXR acetylation by HA-p300 in vivo, immunoprecipitates were analyzed by immunoblotting using the acetyl-lysine and PXR antibodies. IP = immunoprecipitation, FLAG = FLAG octapeptide, Ac-CoA = acetyl coenzyme A, AcK = acetyllysine, PCAF = p300/CBP associated factor, HA = hemagglutinin, TSA = Trichostatin A, NAM = nicotinamide, Rif = rifampicin.
Article Snippet: Relative acetylation levels were assessed by Western blot using an anti-acetyllysine antibody (Cell Signaling #9441S).
Techniques: Recombinant, In Vitro, Acetylation Assay, Histone Deacetylase Assay, Transfection, In Vivo, Immunoprecipitation, FLAG-tag