Fig. 3: TRIM21 is the potential E3 ligase for XRCC4 in response to cellular O-GlcNAcylation levels.

A Table showing the candidates of E3 ubiquitin ligases for XRCC4 as predicted by analyzing the interactome of XRCC4. B HCT116 cells were transfected with TRIM28 or TRIM21 siRNAs for 48 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels (n = 3). C HCT116 cells were transfected with FLAG-TRIM28 (n = 4) or Myc-TRIM21 (n = 6) for 24 h. The cell lysates were immunoblotted with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels. D HCT116 cells were transfected with TRIM21 siRNA for 48 h and treated with 20 μM MG132 for 6 h. For the ubiquitination assay, the cell lysates were immunoprecipitated with the indicated antibodies. The polyubiquitination levels of XRCC4 were analyzed by immunoblotting with K48-Ub antibody. E HCT116 cells were transfected with TRIM21 siRNA for 48 h and with OGA for 24 h. The cell lysates were subjected to immunoblotting with the indicated antibodies. XRCC4 expression was normalized to GAPDH levels (n = 3). F HCT116 cells were transfected with OGA for 24 h. For co-immunoprecipitation, the cell lysates were immunoprecipitated with the XRCC4 antibody. The affinity levels between XRCC4 and TRIM21 were analyzed by immunoblotting with the TRIM21 antibody. Co-immunoprecipitated TRIM21 levels were normalized to immunoprecipitated XRCC4 levels (n = 3). B, C, E, F Data were represented as means ± SD. Statistical significance was determined by the two-tailed Student’s t-test or one-way ANOVA for multiple comparisons (**P < 0.01, ***P < 0.001, ns not significant).