Fig. 1: Engineered AMP binding domain enriches AMPylated proteins from E. coli lysates.

A Schematic representation of the AMPylated protein enrichment strategy using GST-hinT H101N immobilized on glutathione agarose as bait to pulldown interacting AMPylated proteins in lysates. Created in BioRender. Sreelatha, A. (2025) https://BioRender.com/6bpqez2. B Protein immunoblotting of proteins enriched using GST-hinT H101N from wild-type E. coli, glnA knockout (ΔGlnA), glnE knockout (ΔGlnE), or SelO knockout (ΔSelO) E. coli lysates. The ponceau-stained membrane is shown. C Protein immunoblots of cell lysates from wild-type E.coli, glnA knockout (ΔGlnA), glnE knockout (ΔGlnE), or SelO knockout (ΔSelO) E. coli. *denotes a non-specific band observed at 70 kDa. The ponceau-stained membrane is shown. D SDS-PAGE analysis of proteins enriched using GST or GST-hinT H101N from E. coli lysates expressing wild-type (WT) E.coli SelO, inactive D256A SelO, or hyperactive V242A SelO. The enriched proteins were visualized by silver stain. E Protein immunoblots of proteins enriched using GST or GST-hinT H101N from E. coli lysates expressing WT SelO, inactive D256A SelO, or hyperactive V242A SelO. The ponceau-stained membrane is shown. Results depicted in Fig. 1B–E are representative of at least 3 independent experiments.