Journal: International Journal of Molecular Sciences
Article Title: Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells
doi: 10.3390/ijms231810300
Figure Lengend Snippet: Identification of HRG as a binding protein to S100A8/A9. ( A ) Recombinant proteins (GST, GST-S100A8, GST-S100A9) were prepared from the E. coli expression system. Ten micrograms of each individual single protein or a preincubated mixture of GST-S100A8 and GST-S100A9 (GST-S100A8/A9) was incubated with human plasma (1 mL) collected from a healthy volunteer donor. After incubation for 30 min at room temperature under gentle shaking, the GST proteins were all pulled down by the addition of glutathione-conjugated Sepharose beads. After the beads were washed extensively, the bound proteins were eluted with glutathione and subjected to SDS-PAGE for analysis. The Coomassie brilliant blue (CBB)-stained gel displayed three clear bands (BP1, 2, and 3) that co-shed with GST-S100A9 and GST-S100A8/A9. ( B ) Interaction of S100A8/A9 with HRG was confirmed by ELISA. A 96-well plate coated with the highly purified human recombinant HRG protein (100 µg/mL) was incubated with the indicated concentrations of biotinylated human recombinant S100A8/A9 protein after blocking with a chemical-based reagent, Blockmaster DB1130, that is very good for quenching the nonspecific binding of S100A8/A9. The binding of S100A8/A9 to HRG was detected by treatment with HRP-conjugated streptavidin and the chemical reaction between the HRP and the substrate used. The background values of S100A8/A9 binding from the wells without HRG coating were deducted. Data are expressed as optical density (O.D.) means ± SD. *** p < 0.001 by Student’s t -test. ( C ) Schematic drawing of the method used to detect the interaction between S100 family proteins and HRG on the HEKT293 cell surface (left). To effectively increase the probability of interaction between S100 proteins and HRG in the extracellular space, all proteins were modified to express in a membrane-anchored form, so that their density was much greater on the narrow cell surface than in the vast extracellular space. HEK293T cells were co-transfected with myc-tagged modified HRG and HA-tagged S100 family-expressing vectors. After the immunoprecipitation of the expressed cell-surface HRG with myc antibody-conjugated beads, the interacting cell surface S100 proteins were detected by the HA antibody (right).
Article Snippet: The antibodies used were as follows: rabbit anti-human S100A8 (Calgranulin A) polyclonal antibody (#sc-20174; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-human S100A9 (Calgranulin B) polyclonal antibody (#sc-20173; Santa Cruz Biotechnology), rabbit anti-human HRG antibody (#PA5-97051; Thermo Fisher Scientific) that cross-reacts with mouse HRG, and mouse anti-tubulin monoclonal antibody (#T5168, clone B-5-1-2; Sigma-Aldrich, St. Louis, MO, USA).
Techniques: Binding Assay, Recombinant, Expressing, Incubation, Clinical Proteomics, Gentle, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Modification, Membrane, Transfection, Immunoprecipitation