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. 2023 Dec 12;14:8217. doi: 10.1038/s41467-023-43001-y

Fig. 2. cGAS promotes TRIM41-mediated ubiquitination and degradation of ORF2p.

Fig. 2

a, b cGAS promotes ORF2p degradation via the proteasome pathway. Six hours post-transfection with the indicated plasmids, cells were treated with 10 µM MG132 for 12 h, followed by Western blot analysis. c Analysis of ORF2p ubiquitination levels in HeLa cells overexpressing cGAS. Six hours post-transfection of a control vector or cGAS expression vector together with an ORF2p-Flag vector, cells were treated with 10 µM MG132 for 18 h and then lysed for use in co-IP and Western blot experiments. d The effect of knocking out cGAS on the ORF2p ubiquitination level. cGAS WT and knockout cells were transfected with the ORF2p-Flag vector. Then, 6 h post-transfection, the cells were treated with 10 µM MG132 and lysed for use in co-IP and Western blot experiments 24 h post-transfection. e Identification of ORF2p-interacting E3 ligases via mass spectrometry analysis. f Analysis of ORF2p protein levels in cells overexpressing the potential E3 ligases. Vectors encoding HA-tagged PIAS1, TRIM21, TRIM25, TRIM41 or ZNF598 and the ORF2p-Flag expression plasmids were transfected into HEK293T cells, followed by protein extraction 24 h post-transfection. g, h Reciprocal co-IP analysis showed the interaction between TRIM41 and ORF2p in HEK293T cells. i Analysis of the interaction between TRIM41 and ORF2p in vitro with purified HA-tagged TRIM41 and Flag-tagged ORF2p proteins. j, k The effect of TRIM41 overexpression and knockout on ORF2p ubiquitination levels in HeLa cells. l The effect of cGAS overexpression on ORF2p ubiquitination levels in TRIM41-knockout cells. m Western blot analysis of ORF2p protein levels in cells transfected with sgRNAs against TRIM41 and/or vectors encoding cGAS. n Analysis of L1 retrotransposition efficiency in cells transfected with sgRNAs against TRIM41 and/or vectors encoding cGAS. n = 3 independent experiments. Data are presented as mean values ± s.d. One way ANOVA followed by Tukey’s multiple comparisons for (b) and (n). All the Inputs and IPs were from the same experiments. Experiments were repeated three times independently.