Journal: Nature Communications
Article Title: A cysteine selenosulfide redox switch for protein chemical synthesis
Figure Lengend Snippet: Folding and biological activity of biotinylated K1 cyclic analogs. a Primary and tertiary structure of HGF/SF K1 domain (pdb entry 1BHT). b Biotinylated K1 analogs tested for their capacity to bind MET receptor and induce MET-specific phenotypes. The pattern of disulfide bonds determined experimentally corresponds to the native pattern found in K1 domain X-ray crystal structures. c LC-MS monitoring of the folding of cK1-1 peptide into cK1-1f . d Competitive AlphaScreen ® assay with recombinant NK1 protein. K1B or cK1-1f or cK1-2f were mixed with increasing concentrations of NK1 and with extracellular MET domain fused with human IgG1-Fc (MET-Fc) and incubated with streptavidin AlphaScreen ® donor beads and Protein A acceptor beads. Data are presented as normalized percentage of maximal expected signal, i.e. without NK1 competition. Error bars represent the standard deviation (SD) of technical replicates ( n = 3). e MET phosphorylation assay. HeLa cells were treated for 10 min with 300 pM mature HGF/SF (HGF), or with 10 nM/100 nM K1/S , cK1-1f/S , and cK1-2f/S . Cell lysates were then analyzed by specific total MET and ERK or phospho-MET, phospho-Akt and phospho-ERK western blot. Total MET and ERK were used as loading controls after membrane stripping and re-probing. This western blot is representative of two independent experiments ( n = 2). f Cell scattering assay. Capan isolated cell islets were incubated for 18 h in culture media with 300 pM mature HGF/SF (HGF), or 100, 10, 1 nM and 100 and 10 pM K1B , cK1-1f , and cK1-2f . Cell scattering was observed after staining at ×40 (HGF and control, scale bar 500 µm) and ×200 magnification (cK1 treated, scale bar 100 µm). These micrographs are representative of two independent experiments ( n = 2).
Article Snippet: Cell lysates were then analyzed by western blot using specific total MET (#37-0100 Invitrogen), total ERK2 (#SC-154 Tebu-bio), phospho-MET (Y1234/1235, clone CD26, #3077 Cell Signaling), phospho-Akt (S473, clone CD9E, #4060 Cell Signaling), phospho-ERK (T202/Y204, clone E10, #9106 Cell Signaling).
Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Amplified Luminescent Proximity Homogenous Assay, Recombinant, Incubation, Standard Deviation, Phosphorylation Assay, Western Blot, Stripping Membranes, Scattering Assay, Isolation, Staining