Journal: PLOS One

Article Title: Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network

doi: 10.1371/journal.pone.0355964

Figure Lengend Snippet: (A) SPR analysis of Gal-9 binding kinetics to PD-1, TIM-3, CD28, and B7-H4. Human PD-1, TIM-3, and CD28 were fused to the same mouse IgG2a Fc tags. 1.2 µg/mL Gal-9 was injected over a Protein A/G-coated Biacore chip to assess binding kinetics to each immobilized Fc-fusion protein. (B) Flow cytometry analysis of the binding between the four proteins (shown in Panel A) and 293T cells transiently overexpressing either WT Gal-9 or the Gal-9 R65A mutant. ( C ) Cell-surface binding of CD28 to Gal-9 analyzed by Co-IP-Western blotting. Cell-surface expression of CD28 on Jurkat T cells was examined by flow cytometry (left panel); CD28 expression was detected using an anti-CD28 antibody (red), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel); 400 µL of whole cell lysates (WCL) from Jurkat T cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-CD28 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-CD28 antibody from that used for IP, together with an anti-Gal-9 antibody, as indicated. ( D ) Cell-surface binding of B7-H4 to Gal-9 was analyzed by Co-IP-Western blotting. Cell-surface expression of B7-H4 on 293T cells transiently transfected with B7-H4 (293T-B7-H4) was examined by flow cytometry (left panel); B7-H4 expression was detected with an anti-B7-H4 antibody (blue), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel), 400 µL of WCL from 293T-B7-H4 cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-B7-H4 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-B7-H4 antibody from that used for IP, and an anti-Gal-9 antibody, as indicated. Anti-GAPDH antibody was used as an internal loading control for Western blotting of WCL samples in (C) and (D). (E) Gal-9-mediated stimulation of pCD28 signaling activity in Jurkat T cells. Jurkat T cells were treated with or without plate-bound B7.1, and Gal-9’s dose-dependent effects on pCD28 were analyzed by western blotting. Fold change (FC) in pCD28 band intensity relative to the CD28 loading control was quantified using ImageJ and GraphPad Prism.

Article Snippet: Western blotting was then performed as described above using an anti-CD28 rabbit monoclonal antibody (ABclonal, A20346), different from that used for immunoprecipitation, and an anti-Gal-9 mouse monoclonal antibody (R&D Systems, MAB20455) as primary antibodies, followed by appropriate HRP-conjugated secondary antibodies: HRP-anti-Rabbit IgG antibody (Jackson ImmunoResearch, 111-035-003), HRP-anti-mouse IgG antibody (Jackson ImmunoResearch, 115-035-003), and HRP-anti-GAPDH antibody (ABclonal, AC035).

Techniques: Binding Assay, Injection, Flow Cytometry, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Incubation, Transfection, Activity Assay