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. 2021 Mar 22;3(1):zcab010. doi: 10.1093/narcan/zcab010

Figure 2.

Figure 2.

TRDMT1 is ubiquitinated by E3 ligase TRIM28 at sites of DNA damage. (A) GFP-TRDMT1 stably expressed 293 cells were immunoprecipitated by anti-GFP and washed with sodium chloride concentrations at 150 or 300 mM. E3 ligases identified from mass spectrometry by pulling down by GFP-TRDMT1 under each wash condition and numbers of unique peptides of E3 ligases are indicated. (B) 293 TRDMT1 KO cells stably expressing GFP-tagged TRDMTWT and TRDMT1G155Vwere pulled with anti-GFP and immunoblotted with anti-ubiquitin. Cells were treated with 20 μM MG132 for 6 h before pulling down. (C) IP of GFP-TRIM28 and TRDMT1. GFP-vector or GFP-TRIM28 transfected U2OS-TRE cells were treated with or without IR. GFP-vector or GFP-TRIM28 was pulled down by anti-GFP and immunoblotted with anti-TRDMT1. (D) Schematic diagram of KillerRed (KR)-mediated damage at genomic loci in U2OS-TRE cells. (E) U2OS-TRE cells co-transfected TA-KR/TA-Cherry/tetR-KR/tetR-Cherry with or without GFP-TRDMT1 plasmid were exposed to light for 30 min for KR activation and allowed to recover for 30 min before fixation. Recruitment of TRIM28 with anti-TRIM28 antibody at each indicated site is shown. (F) Quantification of the frequency of cells in 50 cells with GFP-TRDMT1 or TRIM28 foci from three independent experiments (n = 3, mean ± SD). (G) WB of TRDMT1 and TRIM28 and the survival rate of WT cells and TRDMT1 KO U2OS cells with or without siTRIM28 after 2 Gy IR treatment.