Fig. 6: Validation of Nt-arginylation candidate proteins using R-catcher pulldown assay.
From: Implementing N-terminomics and machine learning to probe Nt-arginylation
a A schematic representation describing the R-catcher pulldown assay for validation of Nt-arginylation candidate proteins. b R-catcher pulldown assay of Nt-arginylation candidate proteins in HeLa cells treated with MG132 10 μM plus thapsigargin 50 nM. HSPA5 and CALR are positive controls showing that the assay system works properly. P.C. positive control. c List of proteins tested for R-catcher pulldown assay. Position: residue number in the protein sequence where Nt-arginylation occurs; Arginylation site: 10 amino acid sequence surrounding the Nt-arginylation site, spanning 5 residues on each side; Cleavage type: proteases specified by the motif of Nt-arginylation sites. d Dipeptide competition R-catcher pulldown assay to validate the binding is Nt-arginylation-dependent. HeLa cell lysates stimulated with MGTG were incubated with a purified R-catcher in the presence of 25 mM dipeptides, RA or AR. e R-catcher pulldown assay to validate ATE1-dependent arginylation of candidate proteins using Ate1+/+ and Ate1−/− mouse embryonic fibroblasts. P.C. positive control. The bands of immunoblot are representatives of two (e) or three independent experiments (b, d).
