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. 2022 Mar 22;13(4):8000–8012. doi: 10.1080/21655979.2022.2048991

Figure 4.

Figure 4.

UBE2B promotes RAD18 mediated ZMYM2 monoubiquitination and stabilization. (a and c). QRT-PCR (a) and western blotting (b) results showing the expression of ZMYM2 at the mRNA and protein levels in CAOV4 cells 48 h after lentiviral mediated flag-UBE2B overexpression. (b and d). QRT-PCR (b) and western blotting (d) assays to check the expression of ZMYM2 at the mRNA and protein levels 48 h after lentiviral mediated UBE2B knockdown in OVCAR3 cells, with or without the treatment of MG132 (10 μM, 10 h). (e and g) Western blotting assays to detect the expression of UBE2B, RAD18, and ZMYM2 in CAOV4 cells 48 h after lentiviral mediated flag-UBE2B overexpression alone or combined with RAD18 knockdown. Additional MG132 treatment (10 μM, 10 h) was administrated in panel G. (f and g)Western blotting assays to detect the expression of UBE2B, RAD18, and ZMYM2 in OVCAR3 cells 48 h after lentiviral mediated UBE2B knockdown alone or combined with RAD18 overexpression in OVCAR3 cells, with (h) or without (f) the treatment of MG132 (10 μM, 10 h). (i) Co-IP assays to detect the ubiquitination status of ZMYM2 in CAOV4 cells with indicating selective combinations of HA-Ub overexpression, flag-UBE2B overexpression, ZMYM2 overexpression, and RAD18 knockdown. Immunoprecipitation was performed using rabbit anti-myc tag (1:50, 16286-1-AP). Ubiquitinated ZMYM2 was detected using mouse anti-HA tag (66006-2-Ig). (j–l) Representative images (j) and quantitative analysis (k-l) of CHX chase assay in CAOV4 cells with RAD18 knockdown alone, UBE2B overexpression alone, or in combination. 24 h after lentiviral infection, cells were treated with 50 μg/mL of CHX for indicated times to inhibit protein synthesis. Quantitative results were generated by using the ImageJ software. ZMYM2 expression was normalized to that of β-actin. The normalized ZMYM2 expression at 0 h was defined as 1.0 for each panel. n = 3 per group. *, comparison between shNC and shRAD18 or between vector and UBE2B groups. #, comparison between UBE2B and shRAD18+ UBE2B groups. n.s., not significant; ** and ##, p < 0.01; *** and ###, p < 0.001. Scale bar: 20 μm.