p-src y418 antibody  (Thermo Fisher)

Journal: Developmental cell

Article Title: Arp2/3 complex is required for macrophage integrin functions but is dispensable for FcR phagocytosis and in vivo motility

doi: 10.1016/j.devcel.2017.08.003

Figure Lengend Snippet: A) Scanning electron micrograph of WT and Arpc2−/− macrophages with 10 micron C3bi-coated beads. Scale = 1 µm. B) WT and Arpc2−/− macrophages at early (20 min.) and later (60 min.) time points bound to 6 micron beads. Staining (L to R): F-actin, external beads, all beads. Arrows denote internalized beads. Scale = 5 µm. C) Phagocytic index (internalized beads/total beads) of WT (black bars) or Arpc2−/− (KO, grey bars) macrophages at early (20 min.) or later (60 min.) time points with 2, 6, or 10 micron C3bi-opsonized beads. Data is plotted as mean ± SEM. **p = 0.0063, *p ≤ 0.0258. Means represent compiled data from three separate experiments. D) Phagocytic index (internalized beads/total beads) of WT macrophages acutely treated with DMSO (−, black bars) or 150 µM CK-666 (+, grey bars) at early (20 min.) or later (60 min.) time points with 2, 6, or 10 micron C3bi-opsonized beads. Data is plotted as mean ± SEM. ***p = 0.0005, **p = 0.0028, *p < 0.05. Means represent compiled data from three separate experiments. E) Flow cytometry analysis of αM and β2 integrin surface levels in WT (black bars) and Arpc2−/− (KO, grey bars) macrophages. Average Mean Fluorescence Intensity (MFI) of αM and β2 integrin on cultured macrophages were plotted ± SEM. Data were combined from three independent datasets. Negative control (−) cells were incubated with secondary antibody only. No statistically significant p-values were detected. A.U., Arbitrary Units. F) αM and β2 integrin surface levels on spread WT (black bars) and Arpc2−/− (KO, grey bars) macrophages in culture plated on 1 µg/mL collagen. Mean integrated pixel densities are plotted ± SEM. No statistically significant p-values were detected. A.U., Arbitrary Units. N = at least 132 cells. G) Confocal images (L to R, with insets) of αM integrin, F-actin and p34 in WT (top row) or Arpc2−/− macrophages (bottom row) plated on 1 µg/mL collagen. Scale = 20 µm. H) Scanning electron micrograph of WT or Arpc2−/− macrophages with bound C3bi-opsonized 2 micron beads. Scale = 500 nm. See also Figure S2.

Article Snippet: Reagents Drugs : CK-666 (Sigma), Cytochalasin D (Sigma), Blebbistatin (Sigma), Y-27632 (Sigma); Antibodies : Arpc2 (Millipore), Arp2 (ECM Bio), Arp3 (clone FMS338, Sigma), GAPDH (clone 6C5, Ambion/Life technologies), actin (Clone C4, Millipore), Vinculin (clone hvin1, Sigma), CD11b/aM integrin (Abcam), b1 integrin (Thermo), b2 integrin (Novus), p-Y418 Src Family Kinase (Cell Signaling), p-Tyr (4G10, EMD Millipore), Myosin IIA (Abcam), Myosin IIB (Cell Signaling), p-Erk 1/2 (Cell Signaling), C3 (Abcam), FcγRII/III (Fisher), Src-Family Kinases (Cell Signaling); Chemicals : 4-hydroxy-tamoxifen (Sigma), Tamoxifen (Sigma), Other : Phalloidin was conjugated to Alexa 488 or Alexa 568 for all experiments (Life Technologies), pHrodo red-avidin (Thermo), biotin-IgG (Rockland), EZ-Link NHS-LC-Biotin (Fisher).

Techniques: Staining, Flow Cytometry, Fluorescence, Cell Culture, Negative Control, Incubation

Journal: Developmental cell

Article Title: Arp2/3 complex is required for macrophage integrin functions but is dispensable for FcR phagocytosis and in vivo motility

doi: 10.1016/j.devcel.2017.08.003

Figure Lengend Snippet: A, C, E) Wind rose plots generated from WT or Arpc2−/− macrophages migrating in a CSF gradient (A), CX3CL1 gradient (C) or FN gradient (E). Each plot is from one representative experiment of mixed WT and Arpc2−/− cells. 0°on the rose plot is the direction of the gradient. B, D, F) Forward Migration Index (FMI) of WT and Arpc2−/− macrophages plotted as mean ± 95% CI in CSF (B), CX3CL1 (D), or FN gradients (F). Data tables relate mean ± SEM of cell speed (V), FMI, d/T and total cells tracked (N). Quantitative measurements were pooled from multiple experiments using identical chamber conditions. G) WT and Arpc2−/− macrophages plated on 1 µg/mL collagen stained for αM integrin, vinculin and p-Tyr (4G10), β1 integrin, WAVE2 and F-actin at bound 6 micron Cy5-fibronectin (Cy5-FN) beads after 15 minute incubation. Arrowheads denote bound beads on Arpc2−/− macrophages. Scale = 10 µm. H) Localization of β1 integrin, p-Tyr and F-actin to Cy5-FN beads 5, 15 or 30 minutes after bead binding by WT or Arpc2−/− macrophages plotted as mean integrated pixel density (A.U.) ± SEM. **p = 0.0011, ***p < 0.0001. I) Schematic depiction of integrin-Arp2/3 complex coordination. Upon ligation integrin initiates an outside-in signal (black arrows) that induces the Arp2/3 complex to nucleate a branched actin network (an inside-out response). The branched actin network then reinforces the ‘upstream’ pathway (red arrows), perhaps by coordinating or concentrating these factors at new sites of integrin engagement. Without the Arp2/3 complex, macrophages still produce an outside-in signal but lack the ability to reinforce the initial signal, leading to loss of ECM sensing and inhibition of CR3 phagocytosis. See also Figure S6, Movies S4 and S5.

Article Snippet: Reagents Drugs : CK-666 (Sigma), Cytochalasin D (Sigma), Blebbistatin (Sigma), Y-27632 (Sigma); Antibodies : Arpc2 (Millipore), Arp2 (ECM Bio), Arp3 (clone FMS338, Sigma), GAPDH (clone 6C5, Ambion/Life technologies), actin (Clone C4, Millipore), Vinculin (clone hvin1, Sigma), CD11b/aM integrin (Abcam), b1 integrin (Thermo), b2 integrin (Novus), p-Y418 Src Family Kinase (Cell Signaling), p-Tyr (4G10, EMD Millipore), Myosin IIA (Abcam), Myosin IIB (Cell Signaling), p-Erk 1/2 (Cell Signaling), C3 (Abcam), FcγRII/III (Fisher), Src-Family Kinases (Cell Signaling); Chemicals : 4-hydroxy-tamoxifen (Sigma), Tamoxifen (Sigma), Other : Phalloidin was conjugated to Alexa 488 or Alexa 568 for all experiments (Life Technologies), pHrodo red-avidin (Thermo), biotin-IgG (Rockland), EZ-Link NHS-LC-Biotin (Fisher).

Techniques: Generated, Migration, Staining, Incubation, Binding Assay, Ligation, Inhibition