Fig. 5: KRASMut lysine 104 is acetylated by the p300 acetyltransferase.
From: Oncogenic KRAS mutation confers chemoresistance by upregulating SIRT1 in non-small cell lung cancer
A HEK293T cells were transfected with KRASWT, Flag-E.V., and Flag-p300 plasmids. Cell lysates were immunoprecipitated with IgG and an anti-KRAS antibody and then immunoblotted with anti-acetyl-lysine, anti-Flag, and anti-KRAS antibodies. B, C Myc-His-KRASG12C, Flag-SBP-E.V., Flag-SBP-p300, siCon, and/or sip300 were overexpressed in HEK293T cells; after 48 h, cell lysates were separated into nuclear and cytoplasmic fractions, which were subjected to immunoprecipitation with an anti-KRAS antibody and immunoblotting with anti-acetylated lysine and anti-KRAS antibodies. TATA-binding protein was used as a nuclear marker, and tubulin was used as a cytoplasmic marker to verify that nuclear separation was successful. D HEK293T cells were transfected with GFP-E.V., GFP-KRAS (all lysine residues between a.a. 101 and 147 except for K104 mutated to arginine), Flag-SBP-E.V., Flag-SBP-p300, siCon, and/or sip300. Protein lysates were immunoprecipitated with an anti-GFP antibody and then immunoblotted with anti-acetylated lysine, anti-KRAS, and anti-GFP antibodies. E The recombinant KRASG12C protein and the p300 C.D. (catalytic domain, aa 1284–1674, 45.1 kDa) were incubated in the acetylation assay reaction mixture at 32 °C for 4 h, and lysine-acetylated peptides were then identified using an anti-acetyl-lysine antibody. F Lysine-acetylated KRASG12C peptides were also incubated with recombinant SIRT1 protein in the deacetylation assay reaction mixture for 4 h. Deacetylation was quantified using an anti-acetyl-lysine antibody.
