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. 2021 Sep 7;12(5):e02316-21. doi: 10.1128/mBio.02316-21

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Copyright © 2021 Song et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 3 — SdjA interferes with the ubiquitin ligase activity of SdeB and SdeC but not SidE or SdeA. (A) HEK293 cells were transfected to express GFP-tagged SidE family effectors together with mCherry-tagged SdjA and 3×HA-Ub-AA for 18 h. Total proteins resolved by SDS-PAGE were probed with the HA antibody to evaluate ubiquitination induced by SidEs. The expression of SdjA (by mCherry-specific antibodies) and the testing of SidE family effectors (by GFP-specific antibodies) were examined. GAPDH was probed as a loading control. Note that SdjA did not interfere with the expression of SdeB or SdeC but effectively inhibited ubiquitination induced by these proteins. (B) Rab33b ubiquitination induced by SdeB and SdeC is inhibited by SdjA. HEK293 cells were transfected with plasmids that direct the expression of Flag-Rab33b, SdjA, and HA-tagged members of the SidE family. Ubiquitination of Rab33b was indicated by the increase of its molecular weight in immunoblotting with the Flag antibody. The expression of relevant proteins was detected with appropriate antibodies. Data shown are from one representative of three independent experiments with similar results. (C) Diagram of the experimental procedure used to determine the impact of SidJ or SdjA on the activity of the mART domains from members of the SidE family. HA-tagged mART domains from members of the SidE family were coexpressed with GFP, GFP-SidJ, or GFP-SdjA in mammalian cells by transfection. The mART domains isolated by immunoprecipitation with agarose beads coated with HA-specific antibody were incubated with NAD and ubiquitin to produce ADPR-Ub, followed by modification of Rab33b with the SdeA_E/A mutant. SidJ with the ability to inhibit the activity of all members of the SidE family was used as a control. (D) The mART domain of SdeB (or SdeC) coexpressed with SdjA lost the activity to produce ADPR-Ub and thus was unable to ubiquitinate Rab33b with SdeA_E/A. (E) Coexpression of the mART domain of SdeA or SidE with SdjA did not affect their ability to produce ADPR-Ub. Note the production of Ub-Rab33b with SdeA_E/A. In contrast, SidJ inactivates such activity.