SET7/9 methylates TIP60 in vitro and in vivo. (A) Purified GST-TIP60 was incubated overnight at 30°C with increasing amounts of GST-SET7. Reaction mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained by Coomassie or exposed to an autoradiographic film. (B) SET7 was overexpressed in HCT116 cells. IPs using anti-methyl lysine antibody were performed. Methyl lysine levels were normalized by input of TIP60. (C) HCT116 cells were treated with 1 uM SET7 inhibitor (R)-PFI-2 for 24 h. IPs using an anti-methyl lysine antibody were performed. Methyl lysine levels were normalized by input of TIP60. (D) Purified GST-TIP60 deletion mutants were incubated overnight at 30°C with GST-SET7. Reaction mixtures were separated by SDS-PAGE and analyzed via a phosphorimager. (E) Mass spectrometry analysis (LC-MS/MS) was performed, and methylation status of the K137 residue was determined. Purified GST-TIP60Δ2 or GST-TIP60Δ2 point mutants were incubated overnight at 30°C with GST-SET7. Reaction mixtures were separated by SDS-PAGE and analyzed via a phosphorimager. (F) HEK293T cells were transfected with the indicated plasmids and IPs using an anti-methyl lysine antibody were performed. Methyl lysine levels were normalized by input of TIP60. (G) pcDNA3.1-SET7 and Flag-TIP60 were overexpressed in HCT116 cells. The cell lysates were immunoprecipitated with an anti-SET7 antibody. Associated proteins were eluted, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted using the indicated antibodies.
