Fig. 6: TRIM65 promotes the ubiquitination and degradation of TOX4. | Cell Death & Disease

Fig. 6: TRIM65 promotes the ubiquitination and degradation of TOX4.

From: E3 ligase TRIM65 alleviates intestinal ischemia/reperfusion injury through inhibition of TOX4-mediated apoptosis

Fig. 6

A HEK293T cells were co-transfected with GFP-TOX4 and HA-Ub plasmids plus Flag-TRIM65 or Flag-TRIM65 mutant and the cells were treated with MG132 for 4 h. After immunoprecipitation by anti-GFP antibody, ubiquitination level of TOX4 was detected by western blot. B HEK293T cells were co-transfected with GFP-TOX4 and HA-Ub plasmids plus Flag-TRIM65 or not and the cells were treated with MG132 for 4 h. The different type of ubiquitination of TOX4 was detected by western blot using the K48 or K63 specific antibody. C HEK293T cells were co-transfected with GFP-TOX4 and Flag-TRIM65 plasmid plus HA-Ub-K48R or HA-Ub-K63R and the cells were treated with MG132 for 4 h. Ubiquitination level of TOX4 was detected by western blot after immunoprecipitation by anti-GFP antibody. D HA-TOX4 plasmid was transfected to IEC-6 cells with or without GFP-TRIM65 and the cells were treated with MG132 for the indicated time. E Intestinal epithelial cells were treated with CHX plus DMSO or MG132 for indicated times. Expression of TOX4 protein in intestinal epithelial cells in each group was detected by western blot. F Quantification of the bands was carried out using Gel-Pro Analyzer software. All results are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey’s test.

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