Fig. 4: hinT mutants display varying specificity and sensitivity to AMPylated substrates.

A Representative dot blot assay of serial dilutions (400 ng to 25 ng) of proteins spotted on nitrocellulose membranes and probed with GST, GST-Ec hinT, GST-Ec hinT^E96S, GST-Hs hinT or GST-Tm hinT. Bound proteins were visualized using α-GST immunoblotting. Results are representative of at least 3 independent experiments (Supplementary Fig. 9). B α-AMP immunoblotting of enriched proteins from pulldown assay of GST, GST-Ec hinT, GST-Ec hinT^E96S, GST-Hs hinT or GST-Tm hinT with AMPylated GlnA, SodA, Rab1 and Rac1. Results are representative of at least 3 independent experiments (Supplementary Fig. 10b–e). C α-AMP immunoblotting of sucA-AMP bound to GST, GST-Ec hinT, GST-Ec hinT^E96S, GST-Hs hinT or GST-Tm hinT in the presence of increasing concentrations of AMP or GMP nucleotides. Results are representative of at least 3 independent experiments (Supplementary Fig. 11). D Coomassie blue staining analysis of AMPylated sucA purified using GST-Tm hinT affinity. Protein immunoblots for α-AMP and α-his are shown. Results are representative of at least 3 independent experiments. Source data are provided as a Source Data file.