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. 2022 Oct 21;13(10):889. doi: 10.1038/s41419-022-05231-8

Fig. 7. RNF5 promoted STING ubiquitination and degradation.

Fig. 7

A NRCMs were infected with different amounts of AdFlag-RNF5, and the protein expression of STING was detected. B NRCMs infected with AdFlag-RNF5 or AdNull were treated with MG132 or chloroquine (CQ) of 25 μM for 6 h before harvest and the protein expression of STING was detected. C HEK-293T cells were transfected with Flag-STING, Myc-Ub, and HA-RNF5 plasmids, and the ubiquitination of STING was analyzed by WB. D Ubiquitination of STING by RNF5 with the ubiquitin mutants (K48O or K48R). E HEK-293T cells were transfected with Flag-STING (WT), Flag-STING (K20R), Flag-STING (K137R), Flag-STING (K150R), Myc-Ub, and HA-RNF5 plasmids, and the ubiquitination of STING was analyzed by WB. F After HEK-293T cells were transfected with Flag-RNF5 (WT) and Flag-RNF5 (C42S) plasmids, the protein expression of STING was detected. G After HEK-293T cells were transfected with HA-STING, Myc-Ub, Flag-RNF5 (WT), and Flag-RNF5 (C42S) plasmids, MG132 was added 6 h before harvest, and the ubiquitination of STING was analyzed by WB. H RT-PCR analysis of the mRNA levels of ANP, BNP, MYH7, and MYH6 in NRCMs infected with AdVector, AdFlag-RNF5, or AdFlag-STING after PE treatment for 24 h. I WB analysis of RNF5, ANP, BNP, and MYH7 in NRCMs infected with AdVector, AdFlag-RNF5, or AdFlag-STING after PE treatment for 24 h. J NRCMs were infected with AdVector, AdFlag-RNF5, or AdFlag-STING, and then NRCMs were treated with PE for 24 h. Cardiomyocytes were identified by α-actinin staining (red) and nuclei were stained with DAPI (blue). Scale bars: 20 μm. Statistical results for the cell surface area of cardiomyocytes in different groups (n = 40 cells per group). For (H, J) *P < 0.05 or **P < 0.01 versus AdVector AdVector PE, ##P < 0.01 versus AdFlag-RNF5 AdVector PE. Data were presented as the mean ± SD. Statistical analysis was carried out by one-way ANOVA.