Journal: JCI insight

Article Title: α2,6 Sialylation mediated by ST6GAL1 promotes glioblastoma growth.

doi: 10.1172/jci.insight.158799

Figure Lengend Snippet: Figure 4. ST6GAL1 targeting decreases levels of a subset of N-glycoproteins that are known BTIC regulators. (A) Schematic of proteomic analysis of D456 BTICs with and without ST6GAL1 KD (n = 4 for each group of shNT, sh32, and sh33). IB with samples independent of the proteomic analysis verified that suc- cessful targeting ST6GAL1 resulted in decreased (B) PDGFRB, (C) ALCAM, and (D) NRP1 protein. (E) Schematic of pulldown using SNA-bound Agarose beads. (F) SNA pulldown and protein A/G bound agarose beads as a control demonstrated that PDGFRB, ALCAM, and NRP1 were targets for α2,6 sialylation. (G) SNA pulldown of D456 PDX cells with ST6GAL1 KD compared with NT, illustrating differential pulldown of PDGFRB. (H) PDGF-BB–induced (10 minutes) activation of PDGFRB in D456 GBM PDX cells with ST6GAL1 KD compared with NT; IB for p-PDGFRB and total PDGFRB. The experiments were repeated in at least 3 independent biological replicates. Data from 1 representative experiment are shown.

Article Snippet: The primary Abs for Western blot were ST6GAL1 (catalog AF5924, R&D Systems), SOX2 (catalog 561469, BD 1 2 R E S E A R C H A R T I C L E JCI Insight 2022;7(21):e158799 https://doi.org/10.1172/jci.insight.158799 Biosciences), Tubulin (catalog ab21058, Abcam), PDGFRB (catalog 3169, Cell Signaling), Phospho-PDGFRB (Tyr751) (catalog 3161, Cell Signaling), NRP1 (catalog AF3870, R&D Systems), and ALCAM (catalog AF656, R&D Systems).

Techniques: Control, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CD318 is a ligand for CD6

doi: 10.1073/pnas.1704008114

Figure Lengend Snippet: CD6 interacts with CD318. (A) HT1080 CD166 KO cells are deficient of CD166. The CD166 KO cells were analyzed for CD166 expression by flow cytometry. Thin line, isotype control; thick line, CD166 KO cells stained with an anti-CD166 mAb; thin dash line, WT HT1080 cells stained with the anti-CD166 mAb. Data are representative of five independent experiments. (B) HT1080 CD166 KO cells express CD318. The CD166 KO cells were analyzed for CD318 expression by flow cytometry using the anti-CD318 mAb. Thin line, isotype control; thick line, CD166 KO cells stained with the anti-CD318 mAb. Data are representative of five independent experiments. (C) CD6 binds to WT and CD166 KO cells. WT and CD166 KO cells were incubated with the same concentrations of human IgG1 (control) or recombinant human CD6-Fc fusion protein (1 µM), followed by detecting the cell surface-bound CD6 using an Alexa488-anti-human IgG Ab. Thin line, isotype control; thick line, CD166 KO cells stained with CD6; thin dashed line, WT cells stained with CD6. Data are representative of six experiments. (D) Binding of CD6 onto CD166 KO cells is competitively inhibited by soluble CD318. CD166 KO cells were incubated with recombinant human CD6-Fc fusion protein (1 µM) in the presence of different concentrations of recombinant soluble CD318 (0, 1, and 3 µM), then level of cell surface-bound CD6 was quantitated by flow cytometric analysis after staining the cells with an Alexa488-anti-human IgG Ab. Thin shaded line: isotype control; thick dash line, no rCD318; thin dash line, 1 µM CD318; thin line, 3 µM rCD318. The numbers in parentheses represent the molar concentrations of either rHCD6 or rCD318. Data are representative of six independent experiments. (E) CD6 immunoprecipitates CD318 from the CD166 KO cell lysates. CD166 KO cell lysates were immunoprecipitated with the same concentrations of either soluble CD6 or human IgG1, then the immunoprecipitates were separated by SDS/PAGE and probed with the anti-CD318 Ab, showing that CD6 selectively pulled down CD318 (arrow). Data are representative of eight independent experiments. (F) Soluble CD318 binds to CHO cells that express CD6 on the surface. Control CHO cells (solid line) and CHO cells expressing human CD6 (dotted line) were incubated with recombinant CD318. After washing, the binding of CD318 on the cell surface was assessed by flow cytometry after staining the cells with the anti-CD318 mAb.

Article Snippet: Cells lacking cell-surface CD166 were selected by fluorescence-activated cell sorting using an antibody against the extracellular domain of CD166 (R&D Systems; MAB656).

Techniques: Expressing, Flow Cytometry, Control, Staining, Incubation, Recombinant, Binding Assay, Immunoprecipitation, SDS Page

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CD318 is a ligand for CD6

doi: 10.1073/pnas.1704008114

Figure Lengend Snippet: CD318 is a potential biomarker for inflammatory arthritis and chemotactic for T cells. (A) CD318 is highly expressed in synovial tissues from RA patients. Synovial tissue sections from RA, OA, and nonrelevant controls (NL) were stained with either mAb 3A11 (mouse anti-human CD318) or the same amount of control IgGs, then slides were examined under a microscope. (B) Levels of total CD318 are elevated in synovial tissues from RA patients. Synovial tissue from patients with RA (n = 13), OA (n = 20), and normal synovial tissues (Ctrl, n = 17) were homogenized, and levels of total CD318 were analyzed by ELISA. (C) Levels of soluble CD318 are significantly higher in synovial fluids from patients with RA (n = 36) or JIA (n = 10) than in those from patients with OA (n = 28). Sr, serum; SF, synovial fluid. (D) Soluble CD318 is chemotactic to T cells. T-cell migration toward various concentrations of CD318 (50, 100, 200, 400, 800, and 1,600 pg/mL) was assessed in Boyden chemotaxis chambers. PBS was the negative control, and TARC was the positive control. Readings represent the number of cells migrating through the membrane (the sum of three high power 40x fields per well, averaged for each quadruplicate well). (E) T-cell adhesion to IFN-γ–stimulated synovial fibroblasts in the presence of mouse IgG (control), anti-CD318, anti-CD166, or both was measured by a Synergy plate reader.

Article Snippet: Cells lacking cell-surface CD166 were selected by fluorescence-activated cell sorting using an antibody against the extracellular domain of CD166 (R&D Systems; MAB656).

Techniques: Biomarker Discovery, Staining, Control, Microscopy, Enzyme-linked Immunosorbent Assay, Migration, Chemotaxis Assay, Negative Control, Positive Control, Membrane