Fig. 2.

Identification of Net1A acetylation sites. (A) Schematic of Net1A domain structure and acetylation sites. Numbers refer to amino acids in mouse Net1A. (B) Acetylation of endogenous Net1A in MDA-MB-231 cells. Cell lysates were subject to immunoprecipitation (IP) with control or anti-Net1 antibodies. Net1A was tested for acetylation by western blotting. Shown is a representative experiment from two independent experiments. (C) Arginine replacement of the identified acetylated lysine residues prevents Net1A acetylation. HeLa cells were transfected with wild-type Net1A and or Net1A with K83R, K95R, K226R, K227R, K247R and K263R mutations (Net1A 6K/R). Cells were treated overnight with TSA and nicotinamide. Net1A proteins were immunoprecipitated and tested for acetylation by western blotting. Shown is a representative experiment from three independent experiments. (D) Constitutively active Rac1 stimulates Net1A acetylation. HeLa cells were transfected with wild-type Net1A, with or without V¹²Rac1. Net1A proteins were then immunoprecipitated and tested for acetylation by western blotting. Shown is a representative experiment from three independent experiments.