Journal: Journal of Biological Chemistry

Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N-Glycolyl- to N-Acetyl-neuraminic Acid and Generation of Ligands for Siglec-E

doi: 10.1074/jbc.m111.243410

Figure Lengend Snippet: FIGURE 6. Siglec-E-Fc binds subsets of sialylated proteins from activated T-lymphocytes. A, purified T cells were activated for 24 h and either untreated or sialidase-treated and then surface-labeled with biotin. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc com- plexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with streptavidin-HRP to detect surface biotinylated proteins. Arrows indicated proteins that bound to Siglec-E-Fc in a sialidase-sensitive manner (lane 4). B, purified T cells were activated for 24 h and treated or not with sialidase and lysates passed over MAL or SNA affinity columns. Bound sialylated proteins were eluted with SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with siglec-E-Fc precomplexed to anti-Fc-HRP. Arrows indicate proteins that are recognized by siglec-E-Fc in a sialidase-sensitive manner. C, purified T cells were activated for 24 h, cultured for 4 days and treated or not with sialidase. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc complexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with goat anti-CD45 followed by HRP-conjugated anti-goat secondary antibody. The arrow indicates CD45 that is recognized by siglec-E-Fc in a sialidase-sensitive manner. The other bands represent nonspecifically bound proteins.

Article Snippet: For CD45 blots, membranes were blocked as above and then probed with 1 g/ml goat anti-mouse CD45 primary antibody and 1:10000 HRP-conjugated anti-goat IgG secondary antibody (R&D Systems) and detected as described previously.

Techniques: Purification, Labeling, SDS Page, Western Blot, Cell Culture

Journal: Clinical Cancer Research

Article Title: Glioma-Associated Cytomegalovirus Mediates Subversion of the Monocyte Lineage to a Tumor Propagating Phenotype

doi: 10.1158/1078-0432.ccr-11-0414

Figure Lengend Snippet: Figure 1. CMV antigen expression has a tropism for subsets of immune cells within GBMs. A, flow cytometry staining of GBM in single-cell suspension shows that only a subset of cells is positive for the CMV antigen pp65. The percentage of positive cells is denoted in the upper right quadrant of the histogram. B, flow cytometry gating strategy used to distinguish cells of monocyte lineage [such as microglia and Ms (TAMs)] and lymphocytes based on CD11b and CD45 expression profile and intracellular staining of pp65. Each phenotype was separately analyzed for pp65 expression, as indicated by the histograms. The gray line represents the isotype control, and the black line shows the cells with positive staining for pp65. Percentages of cells are denoted in each quadrant and by their respective gates. The percentage of positive cells compared with the isotype control is shown in the right upper quadrant in the pp65 subanalysis panel. These data shown here are representative of the 5 newly diagnosed glioblastoma samples analyzed.

Article Snippet: Working concentrations of appropriate antibodies to surface markers were added [CD133 (MACS), CD45, CD11b, CD3, CD19, CD14 (BD Biosciences), CD163 (R&D), and US28 (Santa Cruz Biotechnology)] and incubated with the cells for 30 minutes in the dark at 4 C. A secondary antibody was required for US28 detection (Invitrogen).

Techniques: Expressing, Flow Cytometry, Staining, Suspension, Control

Journal: Toxins

Article Title: Lebetin 2, a Snake Venom-Derived B-Type Natriuretic Peptide, Provides Immediate and Prolonged Protection against Myocardial Ischemia-Reperfusion Injury via Modulation of Post-Ischemic Inflammatory Response

doi: 10.3390/toxins11090524

Figure Lengend Snippet: Effects of Lebetin 2 (L2) and B-type natriuretic peptide (BNP) on post-ischemic leukocyte infiltration. Myocardial infarction was induced by 35 min of ischemia followed by a 2-day reperfusion period. ( a ) Infiltrated leukocytes were assessed by CD45 immunolabeling in the infarcted area and its border zone (viable area), as well as in the septum (non-ischemic area) of rats treated with saline (1 µL/g), BNP (50 ng/g) or L2 (100 ng/g) intraperitoneally, 5 min before reperfusion. On each section, several fields were photographed (12–30 images/rat, n = 5–6) and the mean number of leukocytes per field was calculated; ( b ) representative microphotographs showing variable density of the leukocytes (CD45 in red and nuclei in blue) in the infarcted area from control and treated animals. Data are mean ± SEM. ***, p < 0.001 vs. saline (control) corresponding group, †††, p < 0.001 vs. corresponding other LV areas. Scale bars = 50 µm. ×20. DAPI, 4′, 6′-diamidino-2-phenylindole.

Article Snippet: Leukocyte infiltration was detected using rat anti-mouse CD45 antibody (1:200; Santa Cruz, Dallas, TX, USA) reacting against most leukocytes.

Techniques: Immunolabeling, Saline, Control