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. 2012 Apr 16;197(2):179–187. doi: 10.1083/jcb.201106152

Figure 4.

Figure 4.

Roles of PIAS4-mediated SUMOylation of HERC2 and RNF168 in cellular DSB responses. (A) Schematic depiction of HERC2 C-terminal fragments (HERC2-C3000) used in B. (B) HEK293T cells transfected with the indicated versions of GFP-HERC2-C3000 for 24 h were exposed to IR and lysed 1 h later. GFP IPs were immunoblotted with the indicated antibodies. MM, molecular mass; WCE, whole-cell extract. (C) U2OS cells were left untreated or transfected with Ubc9-1 siRNA for 48 h, transfected with HERC2 ZZ (residues 2690–2770) constructs for an additional 24 h, and processed as in Fig. 2 A. (D) U2OS cells transfected with siRNAs for 48 h were transfected with the indicated HA-tagged constructs for an additional 24 h, exposed to 4 Gy IR, and fixed 1 h later. Cells were immunostained with HA and 53BP1 antibodies. Arrows indicate cells proficient for 53BP1 accumulation in IR-induced foci. siCTRL, control siRNA. Bar, 10 µm. (E) Quantification of data in D. At least 200 cells were counted for each treatment. Results depict the mean (±SD) of three independent experiments. (F) Cells were treated as in D and coimmunostained with HA and FK2 antibodies. Bar, 10 µm. (G) HeLa/FLAG-SUMO1 cells were transfected with the indicated siRNAs for 72 h and processed as in Fig. 1 C. DOX, doxycycline.