Figure 7: SUMOylation of TARBP2 increases its binding with Ago2.

(a,b) The SUMO-site mutation K52R of TARBP2 affects the interaction between TARBP2 and Ago2. 293T cells were transfected Flag-TARBP2-WT or -K52R with (a) or without (b) myc-Ago2. Forty-eight hours after transfection, cells were lysed for IP with anti-Flag antibody, followed by western blotting with anti-Myc (a) or anti-Ago2 (b) antibody, respectively. (c) SUMOylation enhances TARBP2 binding with Ago2. 293T cells transfected with Flag-TARBP2 were treated with 100 μM H₂O₂ for 3 h before being harvested. Cell lysates were used for IP with anti-Flag antibody, followed by western blotting with anti-Myc antibody. (d) SUMO1 interacts with two SIMs of Ago2. Upper panels: putative SIMs of Ago2 and their mutations (SIM1-wt: ¹⁶²L-D-V-V¹⁶⁵, SIM1-mu: A-A-A-A; SIM-2-wt: ⁵¹⁹V-V-I-L⁵²², SIM2-mu: A-A-A-A. Lower panels: plasmid myc-Ago2-WT, -SIM1-mu or -SIM2-mu with/without GFP-SUMO1 were transfected into 293T cells. IP with anti-Myc antibody and immunoblotting with anti-GFP antibody were performed. (e) Ago2 interacts with SUMO1 conjugated to TARBP2 via its SIMs. pGEX4T1-TARBP2 with or without pE1E2S1 were co-transfected into E. coli BL21 (DE3). GST-TARBP2 protein was purified, and then immunoblotting with anti-SUMO1 and anti-GST antibodies was performed (left panels). This validated that highly SUMOylated GST-TARBP2 was used for pull down of the same amount of each lysate from 293T cells transfected with the control vector, myc-Ago2 WT or SIM1-mu or SIM2-mu, followed by immunoblotting with anti-Myc antibody (right panels). Cell lysates were also used as an Input. (f) The SUMO-site mutation K52R of TARBP2 decreases Ago2. Stable endogenous TARBP2-knocked down 293T cell line (namely TARBP2sh 293 T) was generated by the lentiviral system containing shRNA targeting 3′-UTR of TARBP2 mRNA (left panels). TARBP2sh 293T cells were transfected with Flag-TARBP2-WT or -K52R. Immunoblotting with anti-Dicer, anti-Ago2, anti-Flag and anti-GAPDH antibodies was performed. (g) SUMOylation of TARBP2 stabilizes Ago2. Stable HeLa cell lines expressing Flag-TARBP2-WT or -K52R were treated with 100 μg ml⁻¹ CHX for indicated time and cell lysates were immunoblotted with anti-Ago2, anti-Flag and anti-GAPDH antibodies. For full scans of western blots (a,b,d,g), see Supplementary Fig. 8.