Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Augmentation of human monocyte responses to LPS by the Protein S and Mer/Tyro3 receptor tyrosine kinase axis

doi: 10.4049/jimmunol.1800249

Figure Lengend Snippet: Levels of expression of TAM receptors on monocyte subsets present in human peripheral blood mononuclear cells were determined by labeling with a combination of APC-conjugated CD14 mAb/FITC-conjugated CD16 mAb and PE-conjugated isotype control, Axl, Tyro3, or Mer antibodies prior to flow cytometric analysis. A) Dot plots illustrating the gating strategy to define monocytes on the basis of laser scatter properties (upper panel) and CD14/CD16 antibody reactivity (lower panel). B) Histograms showing binding of antibodies specific for Axl, Tyro3 and Mer, compared with isotype control antibody (grey profile) on CD14++ (red profile) and CD14+/CD16+ (blue profile) monocyte subsets as identified in panel A. C) Quantification of mean fluorescence intensity of staining for antibodies on CD14++ (red bars) and CD14+/CD16+ (blue bars) monocyte subsets. Data shown are mean ± s.e.m., n=4. The difference in mean fluorescence for Mer antibody staining on CD14++ and CD14+/CD16+ monocytes was found to be significant by paired t-test analysis (p<0.01), whereas there was no significant difference for Tyro3 and Axl staining on monocyte subsets.

Article Snippet: Phycoerythrin (PE)-conjugated mouse monoclonal antibodies against human Mer (122518, IgG2b), Tyro3 (96201, IgG1) and Axl (108724, IgG1) together with relevant PE-conjugated isotype controls (IgG1 and IgG2b) were from R&D Systems (Abingdon, UK).

Techniques: Expressing, Labeling, Binding Assay, Fluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Bacillus anthracis Edema Toxin Inhibits Efferocytosis in Human Macrophages and Alters Efferocytic Receptor Signaling

doi: 10.3390/ijms20051167

Figure Lengend Snippet: Monocyte-derived macrophages polarized by dexamethasone (M2(Dex) macrophages) expressed M2 markers and were highly efferocytic. ( A ) The histogram overlays showing the cell surface expression of CD206 and CD163 surface markers on M2(Dex) macrophages (red, M2), unpolarized M0 macrophages (blue, M0), or isotype control antibodies binding to M2(Dex) macrophages (light orange, IC). The mean fluorescence intensities (MFI) ± SEM of 10 independent donors are indicated; ( B ) The gating of efferocytosis by M2(Dex) macrophages shows (left to right) the successive exclusion of un-engulfed apoptotic PMN (FSC vs. SSC gate), the exclusion of macrophage cell surface bound CD66b + PMN, the inclusion of CD163 + M2(Dex) macrophages, and the percentage of CD163 + CD66b − macrophages containing engulfed eFluor670 + PMN. The lower panels show efferocytosis gating. The upper panels depict the staining of cells from the same experiment with the isotype control antibodies for CD66b and CD163; ( C ) M2(Dex) macrophages express MerTK and Tyro3 but not Axl TAM family members. MFI ± SEM of 10 independent donors are indicated. * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison posttest compared to IC.

Article Snippet: Efferocytosis receptor antibodies (R&D Systems) were PE-labeled and included the following specificities and clones: Dtk/Tyro3 (clone 96201), Axl (108724), MerTK (125518), αVβ3 (23C6), or αVβ5 (P5H9).

Techniques: Derivative Assay, Expressing, Binding Assay, Fluorescence, Staining, Comparison

Journal: Journal of Virology

Article Title: Axl Can Serve as Entry Factor for Lassa Virus Depending on the Functional Glycosylation of Dystroglycan

doi: 10.1128/JVI.01613-17

Figure Lengend Snippet: Primary human cells coexpress Axl and differentially glycosylated DG. (A) Detection of candidate LASV receptors in primary human cells. Total cell protein was extracted from primary human hepatocytes (PHHC), HUVEC, HMVEC-L, and SAEC, separated by SDS-PAGE, and blotted onto nitrocellulose. Functional DG was detected with MAb IIH6, which recognizes the matriglycan sugar polymers on α-DG. The presence of the core protein was probed with MAb 8D5 to β-DG, and α-tubulin was included as a loading control. A549 and HT-1080 cells were included as positive and negative controls, respectively. Axl, Tyro3, and DC-SIGN were detected with polyclonal Ab goat anti-human Axl, MAb 96201 anti-human Tyro3, and MAb 120507 anti-DC-SIGN. Human THP-1 monocytes and THP-1-derived immature dendritic cells were used as positive controls for Tyro3 and DC-SIGN, respectively (25). As a negative control for Axl, HEK293H cells were included. The negative-control lane of the Axl blot was taken from the same membrane and moved, as indicated by the thin white line. Primary antibodies were detected with HRP-conjugated secondary antibodies using enhanced chemiluminescence (ECL) for development. The observed differences in the apparent molecular masses of Axl in different cells were consistently observed and may be due to different glycosylation patterns. The expression levels of Axl in PHHC varied between donors, and a representative example was selected. (B) Western blot of WGA-purified DG. Dystroglycan was purified from the indicated cells by WGA affinity chromatography. Concentrated fractions were probed in a Western blot for functional glycosylation of α-DG with MAb IIH6 and for β-DG with MAb 8D5 as described for panel A. (C) Solid-phase virus binding assay. Equal amounts of DG purified from the indicated cells were immobilized in microtiter plates and incubated with the indicated concentrations of purified rLCMV-LASVGP. Bound virus was detected with MAb 83.6 to LASV GP2 using a biotinylated secondary antibody and HRP-conjugated streptavidin in a color reaction. Data are means ± standard deviations (SD) (n = 3).

Article Snippet: Purified polyclonal goat IgG anti-human Axl, MAb 96201 anti-Dtk/Tyro3, and MAb 120507 anti-DC-SIGN were from R&D Systems.

Techniques: SDS Page, Functional Assay, Derivative Assay, Negative Control, Membrane, Expressing, Western Blot, Purification, Affinity Chromatography, Virus, Binding Assay, Incubation