Figure 1. SidJ inhibits the Ub-ADP-ribosylation activity of SdeA.

a) SidJ and SdeA constructs were expressed as indicated in HEK293T cells and ubiquitin modification was probed using the Ub antibodies abcam-Ub and CS-Ub as described previously³. b) Yeast strain W303 was transformed using the indicated combination of constructs. Serial dilutions of transformed yeast were spotted on dextrose (repressing) or galactose (inducing) containing plates. SdeA EE/AA indicates mutation of E860, E862 to alanine. c) Purified SdeA from HEK293T cells expressing SdeA alone or in combination with SidJ was used in ε-NAD⁺ hydrolysis assays. Increase in the fluorescence indicates Ub-ADP ribosylation²⁰. d) SdeA purified from E.coli was incubated with HEK293T cell lysate containing SidJ or depleted of SidJ. SdeA was subsequently purified using glutathione agarose beads and used in ε-NAD⁺ hydrolysis assays. Experiments in panels a-d were repeated thrice independently with similar results. For gel source data, see Supplementary Fig. 1.