Skip to main content
. Author manuscript; available in PMC: 2021 Feb 4.
Published in final edited form as: Cell Rep. 2020 Dec 29;33(13):108569. doi: 10.1016/j.celrep.2020.108569

Figure 5. TRIM41 Acts as a Ubiquitin Ligase for TOP3Bccs and Promotes the Repair of TOP3Bccs.

Figure 5.

(A) Immunoblots showing TRIM41 and R338W-TOP3B expression after TRIM41 downregulation (GAPDH as loading control). HCT116 cells were either transfected with a FLAG-tagged R338W-TOP3B plasmid construct alone or co-transfected with a siTRIM41construct for 72 h.

(B) Immunoblots showing effect of TRIM41 depletion on endogenous TOP3B level. GAPDH was included as loading control. HCT116 cells were transfected with siTRIM41construct for 72 h.

(C) HCT116 cells were transfected with FLAG-tagged WT- or R338W-TOP3Bs or co-transfected with siTRIM41construct for 72 h. TOP3Bcc were detected with anti-FLAG antibody. Equal loading was tested with anti-dsDNA antibody. The figure is representative of two independent experiments.

(D) Quantitation of TOP3Bccs in two independent RADAR assays as shown in (C).

(E) HCT116 cells were transfected with FLAG-tagged R338W-TOP3B alone or with siTRIM41 construct. After 72 h, equal amounts of RADAR assay samples were IPed with anti-TOP3B antibody. IP samples and the input RADAR assay samples were digested with MNase, resolved on SDS-PAGE and immunoblotted with anti-Ub and anti-TOP3B antibodies. Loading of input RADAR samples was tested with anti-dsDNA antibody.

(F) Immunoblots showing TRIM41 and R338W-TOP3B expression after transfection of HCT116 cells with HA-tagged R338W-TOP3B plasmid construct alone or with FLAG-tagged TRIM41. GAPDH served as loading control.

(G) Reduced TOP3Bccs upon TRIM41 overexpression. HCT116 cells were transfected with HA-tagged R338W-TOP3B alone or co-transfected with FLAG-tagged TRIM41 for 48 h. TOP3Bccs were detected with anti-HA antibody. Loading was tested by with anti-dsDNA antibody. The figure is representative of three independent experiments.

(H) Quantitation of TOP3Bcc formation in three independent RADAR assays as shown in (F).

(I) Increased TOP3Bcc ubiquitination upon transfection with TRIM41. HCT116 cells were either transfected with HA-tagged R338W-TOP3B alone or co-transfected with FLAG-tagged TRIM41. After 48 h, RADAR assay samples were IPed with anti-TOP3B antibody. IP samples and the input RADAR assay samples were digested with MNase, resolved on SDS-PAGE, and immunoblotted with anti-Ub and anti-TOP3B antibodies. Loading was tested with anti-dsDNA antibody.

See also Figure S5.