Fig 1: NEDD8, UBA3, UBE2M and RBX1 were over-expressed in AML patients and were correlated with worse OS(A–D) Compared with normal control, the mRNA levels of NEDD8, UBA3, UBE2M and RBX1 were significantly higher in AML patients. (E–G) As revealed by FAB subtype analysis, the mRNA expression levels of NEDD8, UBE2M and RBX1 were higher in patients with AML-M2, M4 and M5 than those in healthy controls. In patients with AML-M5, the mRNA expression of UBA3 was higher than that in the control group. (H–K) Survival analysis revealed that the OS rate of patients with overexpression of NEDD8, UBA3, UBE2M and RBX1 was lower than that of patients with low-expression. ROC curves were plotted based on the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1, respectively, and Jorden index was calculated to determine the cut-off value (NEDD8, 1.4257; UBA3, 1.6511; UBE2M, 1.6146; RBX1, 1.4026) to divide the samples into high and low expression groups. An independent sample t-test was conducted to compare the means between the two groups. Triplicates were set for each gene in each sample for RT-qPCR. Chi-square test was performed to compare the qualitative data. The KM method was employed to draw the survival curves of patients, and log-rank tests were utilized to compare the survival rates. Univariate and multivariate Cox analyses were adopted to determine whether the expression of the interested genes was the independent prognostic factor for OS; ***P<0.001, **P<0.01, *P<0.05.
Fig 2: HUWE1 is the DNA-PKcs neddylation E3.a, b HUWE1 interacts with DNA-PK complex and UBE2M. Immunoprecipitation was performed with DNA-PKcs antibody or Flag-HUWE1, either HUWE1 or DNA-PKcs, Ku70/Ku80, UBE2M was detected by western blotting with indicated antibodies. c HUWE1 depletion abolished DNA-PKcs neddylation. HUWE1 was depleted with specific siRNAs. The depleted cells were harvested and subjected to IP with DNA-PKcs antibody following detection with NEDD8 antibody.
Fig 3: UBA3, UBE2M and RBX1 knockdown inhibited the NEDD8 NEDDylation of CULs and activated the p53 signaling pathway in MOLM-13 cells(A) The knockdown efficiency of shRNA was >70% by RT-qPCR. (B and E) After transfection of shRNA in MOLM-13 cells, the expression of UBA3, UBE2M and RBX1 proteins was reduced. (C) and (F–H) NEDD8 NEDDylation of CUL1-4 was significantly inhibited following the knockdown of UBA3, UBE2M and RBX1. The NEDDylation of CUL5 was suppressed by the knockdown of UBA3 but up-regulated by the knockdown of UBE2M and RBX1. (D) and (I–K) The expression of p21 and p53 significantly increased following UBA3, UBE2M and RBX1; ***P<0.001, **P<0.01. An independent sample t-test was adopted to compare the means between two groups. Each experiment was carried out in triplicate.
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