Fig. 1

AGO2 Is Acetylated at K355, K493 and K720. a, b AGO2 is acetylated. 293T cells transfected with Myc-AGO2 were treated with the deacetylase inhibitors TSA (2 μM) and NAM (10 mM) for 6 h and 18 h before harvested, respectively. Cell lysates were used for IP with anti-Myc antibody or anti-acetylated-Lysine (anti-Ac) or normal mouse IgG (as a negative control), and then AGO2 acetylation was analyzed by Western blotting (WB) with as indicated antibodies. c Single, double and triple mutations of K355, K493 and K720 reduce AGO2 acetylation. The indicated single (left panels), double and triple (right panels) mutations of Myc-AGO2 were transfected into 293T cells. AGO2 acetylation was detected by IP/WB. d TSA/NAM increases the acetylation level of AGO2-WT but not of AGO2-3KR. 293T cells transfected with AGO2-WT or AGO2-3KR were treated with or without TSA/NAM as (a). AGO2 acetylation was analyzed by IP/WB. e TSA/NAM increases AGO2 acetylations at K355, K493 and K720. 293T cells transfected with Flag-AGO2 were treated with or without TSA/NAM as in (a). Cell lysates were IP with anti-Flag antibody, followed by WB with home-made AGO2 specific acetyl-antibodies. f Triple-mutation of AGO2 at K355, K493 and K720 impairs its acetylation. AGO2-WT and AGO2-3KR were transfected into 293T cell, and then AGO2 acetylation was analyzed by IP/ WB with AGO2 specific acetyl-antibodies. See Figure S1A for three acetylation sites K355, K493 and K720 identified by mass spectrometry, Figure S1B-D for the specificity of home-made AGO2 specific acetylation antibodies AGO2-K355-Ac, AGO2-K493-Ac and AGO2-K720-Ac