Figure 8
From: DNA polymerase ι is acetylated in response to SN2 alkylating agents
Characterization of Polι acetylation. (a) Effect of a p300 knock-out on Polι acetylation in cells treated for 1 h with 200 μM MMS or DMS. (b) Effect of a p300/CBP inhibitor, L002, on MMS-induced Polι acetylation. (c) Immunoprecipitation assay on extracts from HEK293T cells co-transfected with FLAG-Polι and wild-type mCBP, or with an L1435A/D1436A (LD) mutation that abolishes acetyltransferase activity of CBP. (d) Effect of 1 h treatment with 200 μM MMS, or DMS, on the cellular level of p300 and CBP. (e) Effect of CBP knock-out on Polι acetylation in cells treated for 1 h with 200 μM MMS or DMS. (f) Effect of p300 overproduction, or MMS, or DMS treatment on Polι or Polη acetylation. Immunoprecipitation of FLAG-tagged polymerases from extracts from HEK293T cells transfected with FLAG-Polι (left panel) or FLAG- Polη (right panel). As indicated, cells were co-transfected with wild-type p300-c-myc, and treated for 1 h with 200 mM DMS, or MMS. Unless otherwise stated, HEK293T cells were transiently transfected with the FLAG-Polι plasmid. 24 hrs post transfection cells were treated for 24 hrs with DKAc inhibitor cocktail. Proteins were visualized by western blot using the following antibodies; polyclonal antibodies against p300 (p300 protein); monoclonal antibodies against acetylated lysines (acetylated Polι); polyclonal rabbit antibodies against a C-terminal fragment of Polι; or monoclonal antibodies against the FLAG epitope, as indicated.
