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. 2020 Sep 8;28(2):671–684. doi: 10.1038/s41418-020-00616-8

Fig. 2. UBE2O negatively controls Mxi1 abundance via the ubiquitin-proteasome pathway.

Fig. 2

a Scramble or UBE2O siRNAs were transfected into lung cancer cells for 48 h. Cells were lysed and then detected by western blotting (n = 3). b A549 and H1299 cells were transfected with indicated siRNAs for 48 h and then irradiated for 1 h. Cells were lysed and then subjected to western blotting (n = 3). p-ATM was used as a positive control upon DNA damage. c ShRNAs-mediated knockdown of UBE2O led to increased Mxi1 protein level (n = 3). d Cells transfected with indicated plasmids for 24 h were collected and then subjected to western blotting (n = 3). e Schematic presentation of wild-type and mutants UBE2O. f HeLa cells were transfected with the indicated plasmids, followed by treatment with MG132 (10 μM) for 4 h prior to collection. The lysates were incubated with S protein beads and then subjected to immunoblotting (n = 3). g HeLa cells were transfected with indicated siRNAs for 24 h, and then co-transfected with SFB-Mxi1 and HA-Ubiquitin plasmids for another 24 h. After treatment with MG132 (10 μM) for 4 h, cells were collected for co-IP with HA beads, and then subjected to immunoblot analysis (n = 3). h Upper panel: Scramble or UBE2O siRNAs were transfected into HeLa cells for 48 h. Cells were then treated with cycloheximide (CHX, 100 μg/mL) and collected for immunoblot analysis at the indicated time points. Lower panel: quantification of Mxi1 band intensity was presented (n = 3). ***P < 0.001.