human gal9 protein  (R&D Systems)

Journal: eLife

Article Title: Galectin-9 regulates the threshold of B cell activation and autoimmunity

doi: 10.7554/elife.64557

Figure Lengend Snippet: Figure 4. Loss of Gal9 leads to enhanced activation and accumulation of B-1a cells at steady-state. (A) Representative plots of IgM expressing cells in the PerC of aged WT and Gal9KO mice, as indicated. (B) Summary proportion of CD5 expressing B-1a cells in the PerC shown in A. (C) Representative histograms of CD86 expression at steady state on PerC cells shown in (A). (D) Summary gMFI of CD86 expression shown in C. (E) Representative plots of ASCs in the PerC of aged mice. (F) Summary proportion of ASCs shown in (E). (G) Anti-phosphorylcholine (PC) specific IgM in sera of aged mice. (H) Representative plots of B-1 cells of IgM expressing cells in the spleen of aged mice. (I) Proportion of B-1 cells shown in H. (J) Representative plots of B-1a and B-1b cells of H. (K) Summary proportion of CD5 expressing B-1a cells in the spleen of aged mice shown in (I). Data are representative of ten biological replicates over three independent experiments. Statistical significance was assessed by Mann–Whitney ****p<0.0001. The online version of this article includes the following figure supplement(s) for figure 4:

Article Snippet: Primary murine B cells were incubated in 20 mM b-lactose or 0.1 mM recombinant Gal9 (R&D Systems) in RPMI1640 at 37 ̊C for 30 min.

Techniques: Activation Assay, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Galectin-9 regulates the threshold of B cell activation and autoimmunity

doi: 10.7554/elife.64557

Figure Lengend Snippet: Figure 5. Gal9 regulates BCR signal transduction in B-1a cells through interactions with IgM and the negative co-receptor CD5. (A) Representative histograms of CD86 expression on B-1a cells from WT (black) and Gal9KO (red) mice stimulated with titrated concentrations of anti-IgM F(ab’)2. (B) Summary proportion of CD86 expressing cells as a function of F(ab’)2 concentration as in (A). (C) EC50 of F(ab’)2 titration shown in (A). (D) Representative histogram of p-Tyr level in B-1a cells from WT (black, open) or Gal9KO (red, filled), and WT B-1a cells treated with lactose (blue, open) and Gal9KO B-1a cells treated with rGal9 (orange, filled) stimulated with anti-IgM (Fab’)2 for 5 min (left), summary gMFI of p-Tyr levels (right). (E) Representative histograms of Gal9 expression in B-1a cells (green), B-1b cells (white), and splenic B-2 cells (purple) from WT mice (left), summary gMFI of Gal9 expression (right). (F) Representative histograms of CD5 detection on rGal9-coated beads incubated with lysates from different B cell subsets, as indicated (left), summary gMFI of CD5 enrichment on beads incubated with lysates (right). (G) Reconstructed dual-dSTORM images of IgM (yellow) and CD5 (cyan) on the surface of B-1a cells from WT and Gal9KO mice, ROI (3 mm x 3 mm) used for analysis is expanded in zoom. Scale bars represent 1 mm. (H) Quantification of IgM mean cluster area from dSTORM images. (I) Quantification of IgM clustering tendency using Hopkins index. (J) Quantification Figure 5 continued on next page

Article Snippet: Primary murine B cells were incubated in 20 mM b-lactose or 0.1 mM recombinant Gal9 (R&D Systems) in RPMI1640 at 37 ̊C for 30 min.

Techniques: Transduction, Expressing, Concentration Assay, Titration, Incubation

Journal: eLife

Article Title: Galectin-9 regulates the threshold of B cell activation and autoimmunity

doi: 10.7554/elife.64557

Figure Lengend Snippet: Figure 6. Gal9 regulates LPS-mediated activation by restraining signal transduction through interactions with TLR4 and the negative co-receptor CD180. (A) Proportion of CD86 expressing cells of B-1a cells from WT (black) and Gal9KO (red) mice stimulated with titrated concentrations of LPS (left), EC50 (right). (B) Titration with CpG-ODN as in A (left), EC50 (right). (C) Titration with Imiquimod as in (A) (left), EC50 (right). (D) Titration with Zymosan as in (A) (left), EC50 (right). (E) Representative histograms of p-Tyr levels at 10 min in B-1a cells from WT (black, open) or Gal9KO (red, filled) mice, or WT Figure 6 continued on next page

Article Snippet: Primary murine B cells were incubated in 20 mM b-lactose or 0.1 mM recombinant Gal9 (R&D Systems) in RPMI1640 at 37 ̊C for 30 min.

Techniques: Activation Assay, Transduction, Expressing, Titration