Fig. 3: Specificity of MavL activity.

A Comparison of (ADP-ribosyl)hydrolysis of ADPR-Ub by MavL, ARH1, and Larg1. Reactions were analyzed by native PAGE and visualized by Coomassie Blue staining. Controls of Ub, ADPR-Ub, and enzymes alone were included. Experiments were performed three times independently with similar results. B Comparison of (ADP-ribosyl)hydrolysis of ADPR-Ub variants by MavL. Reactions were analyzed by native PAGE and visualized by Coomassie Blue staining. Controls of Ub and ADPR-Ub were included. Experiments were performed three times independently with similar results. C In vitro de-PARylation assay for MavL. Purified His-tagged PARP1 was auto-PARylated and incubated with varying concentrations of HsPARG (as a positive control) and MavL. Reactions were immunoblotted against ADPR to show the change in ADP-ribosylation level. Loading of the reactions is shown by immunoblotting against 6 × His-tag. Experiments were performed three times independently with similar results. D In vitro de-MARylation assay for MavL. Purified HisSUMO-tagged PARP10 was auto-MARylated and incubated with varying concentrations of MacroD2 (as a positive control) and MavL. Reactions were immunoblotted against ADPR to show the change in ADP-ribosylation level. Loading of the reactions is shown by immunoblotting against 6 × His-tag. Experiments were performed three times independently with similar results. E–G Specificity of MavL towards other arginine MARylations. Purified MARylated Rab5 (E) and MARylated Gdh2p (F) were incubated with varying concentrations of MavL, whereas immunoprecipitated MARylated ANT1 (G) was incubated with Larg1, MavL, and ARH1. ADP-ribosylation states of the reactions were probed by immunoblotting against ADPR. Loading of the reactions was visualized by Coomassie Blue staining for Gdh2p and Rab5 and by immunoblotting against Flag-tag for ANT1. Experiments were performed three times independently with similar results.