Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: Activated FcγRI is retained in intracellular compartments described by IRAP (A–C) DC2.4 cells expressing FcγRI-GFP (green) were incubated with anti-FcγRI (clone 10.1) and cross-linked with anti-mouse IgG (blue) at 4°C. After removal of excess antibodies, the cells were shifted for the indicated time points at 37°C, fixed and stained for EEA1 (red) (A), IRAP (red) (B) or LAMP1 (red) (C). The pictures show representative images from three independent experiments and the graphs show colocalization between FcγRI and the endocytic markers. Each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Expressing, Incubation, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: Activated FcγRI recruits signaling partners at the level of IRAP endosomes (A–C) DC2.4 cells expressing FcγRI-GFP (green) were incubated with human IgG and anti-human IgG at 4°C. After removal of excess antibodies, cells were shifted for the indicated time points at 37°C, fixed and stained for: pSrc (red) and IRAP (gray) (A), LAT (red) and IRAP (gray) (B), or PLCγ (red) and IRAP (gray) (C). The pictures show representative images from three independent experiments and the graphs show colocalization between the indicated signalosome component and IRAP. Each dot represents a cell. Scale bars = 5 μm. (D) The graph shows the colocalization between FcγRI and IRAP that was quantified in the conditions (A), (B) and (C). (E) The interaction of FcγRI with Syk was measured by FLIM experiments and the graph shows the GFP lifetime for cells expressing only GFP, both GFP and mCherry as soluble proteins and the cells expressing both FcγRI-GFP and Syk-mCherry in steady state and after the receptor crosslinking for indicated time points. In all the graphs, each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Expressing, Incubation, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet: IRAP deletion affects the endosomal recruitment of active Syk to FcγRI (A and B) Wild-type (WT) or IRAP-deficient (KO) BM-DCs were incubated with anti-mouse FcγRI (clone AT152-9), followed by crosslinking with non-labeled anti-rat IgG at 4°C. After removal of excess antibodies, the cells were shifted for the indicated time points at 37°C, fixed and stained for endogenous FcγRI (green) and γ-chain (red) in (A) and for γ-chain (green) and Syk phosphorylated on Tyrosine 352 (pSyk) (red) in (B). The pictures show representative images from three independent experiments and the graphs show colocalization between FcγRI and the γ-chain (A) and colocalization between the γ-chain and phosphorylated, active Syk (B). Each dot represents a cell. Data are represented as mean ± SEM. Scale bars = 5 μm. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Incubation, Labeling, Staining

Journal: iScience

Article Title: Activating FcγR function depends on endosomal-signaling platforms

doi: 10.1016/j.isci.2023.107055

Figure Lengend Snippet:

Article Snippet: Anti-mouse FcγRI , BioRad , Cat# MCA5997; RRID: AB_2687456.

Techniques: Produced, Western Blot, Recombinant, Lysis, Isolation, Enzyme-linked Immunosorbent Assay, Cloning, Software

Journal: ERJ Open Research

Article Title: The effects of exogenous lipid on THP-1 cells: an in vitro model of airway aspiration?

doi: 10.1183/23120541.00026-2016

Figure Lengend Snippet: PMA/THP-1 lipid uptake. PMA/THP-1 cells were incubated with 10% vol/vol Calogen for 24 h with and without inhibitors and were stained with ORO to detect lipid accumulation. a) Untreated cells were compared with b) cells treated with Calogen alone and c) in the presence of cytochalasin D and d) CD36 monoclonal blocking antibody. Inset images are magnified representative regions of each treatment. e) Cells treated with Calogen were also visualised under TEM. Scale bar=1 μm. f, g) Lipid droplets were identified and magnified to reveal dark stained remnants of lipid encased in membrane-bound vesicles, as indicated by the arrows. Scale bars=500 nm (f) and 100 nm (g).

Article Snippet: The plate was centrifuged (350 g , 3 min, 4 °C) and washed twice with PBS before incubating (30 min at 4 °C) with mouse monoclonal primary antibodies (CD11b, CD14, CD16, CD23, CD24, CD32, CD36, CD64, CD80, CD86, CD163, CD206 (20 μg·mL −1 , AbD Serotec, Oxford, UK) or CD32 [ ]).

Techniques: Incubation, Staining, Blocking Assay, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Comparative Characterization of Phosphatidic Acid Sensors and Their Localization during Frustrated Phagocytosis *

doi: 10.1074/jbc.M116.742346

Figure Lengend Snippet: Binding of Spo20p-PABD and Opi1p-PABD to plasma and ER membranes obtained by subcellular fractionation. A, GST or GST-PABDs beads were incubated with plasma (PM) or ER membranes (containing ∼400 μg of unsaturated lipids) for 30 min, and the amount of lipid bound to the beads was estimated using a colorimetric quantification kit for unsaturated lipids. B and C, similar experiments were performed with plasma (PM) and ER membranes previously incubated at 37 °C for 2 h with trypsin or proteinase K, respectively. Proteases were heat-inactivated for 1 h at 75 °C before incubating samples with GST-PABD bound to GSH beads. Results are presented as the means ± S.E. (n = 3) obtained from independent experiments. Significant differences for lipid binding between GST-Spo20p-PABD and GST-Opi1p-PABD are indicated (**, p > 0.01, and ***, p > 0.001). In each condition a background signal measured from GST and GST-PABDs beads alone was subtracted. Inset reveals the absence of the plasma and ER membrane markers (CD64 and calnexin, respectively) as revealed by immunoblotting plasma and ER membranes after trypsin (Try) or proteinase K (PK) treatment as compared with control membranes (50 μg) incubated without protease (Ctrl).

Article Snippet: Anti-histone H3, anti-giantin (Ab24586), anti-Na + /K + -ATPase (Ab58475), anti-HSP-60 (Ab46798), and anti-calnexin (Ab22595) were purchased from Abcam; anti-CD64 (MAB2074) was from R&D Systems; anti-GST (G1160) was from Sigma, and anti-GAPDH (2118S) from Ozyme.

Techniques: Binding Assay, Fractionation, Incubation, Western Blot