p src y418  (Cell Signaling Technology Inc)

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway

doi: 10.1155/2021/6639252

Figure Lengend Snippet: oxLDL regulated expression of toll-like receptor family, ROS-related genes, and sirtuin family as well as activation of Src. After oxLDL stimulation, the mRNA or protein expression of genes was detected by real-time PCR or western blot in VSMCs. The phosphorylation of Src was detected using western blot. The untreated group served as control (Con), and β -actin served as an internal reference gene to normalize protein expression. All of the real-time PCR results were calculated by 2 - ΔΔ CT method; the western blot results were calculated using grayscale value. (a) The mRNA expression of the toll-like receptor family ranging from Tlr1 to Tlr13 . ( n = 3 per group, results were expressed as mean ± SD; ∗ P < 0.01, as compared with the Con group.) (b) Upper: the phosphorylation of Src (Tyr-418, Y418) was detected after 1-hour stimulation by different doses (12.5, 25, and 50 μ g) of oxLDL in VSMCs. Bottom: the protein expression of ROS generation (Nox2 and Nox4) and elimination-related (Mnsod) genes in VSMCs, after stimulating by different doses (12.5, 25, and 50 μ g) of oxLDL for 48 hours. (c) After incubation with different doses (12.5, 25, and 50 μ g) of oxLDL, the protein levels of the sirtuin family (Sirt1-7) were measured. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01 as compared with the Con group).

Article Snippet: Additionally, primary antibodies of β -actin (Cat#3700), p-Src Y418 (Cat#6943), t-Src (Cat#2123), Mnsod (Cat#13141), Sirt1 (Cat#2314), Sirt2 (Cat#12650), Sirt3 (Cat#5490), Sirt5 (Cat#8782), Sirt6 (Cat#12486), Sirt7 (Cat#5360), and tlr4 (Cat#14358) as well as the second antibody-conjunction HRP anti-mouse/rabbit (Cat#7076/Cat#7074) were gained from CST (MA, USA).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Incubation

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway

doi: 10.1155/2021/6639252

Figure Lengend Snippet: Knockdown or inactivation of Src regulated lipid accumulation and cellular phenotype in VSMCs. (a, b) Tlr4(-) or NC were treated with or without oxLDL (50 μ g/mL) for 1 hour; the activation of Src (Tyr-418, Y418) had been detected by western blot. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with oxLDL-untreated NC group; ## P < 0.01, as compared with oxLDL-treated NC group.) Src was knockdown by siRNA transfection within VSMCs. The Src-specific antagonist, PP2, was used to block Src activation, with PP3 serving as the negative control. The VSMCs were treated with and without 50 μ g/mL oxLDL for 48 hours. (c, d) The Src-specific [Src (-)] and negative control (NC) siRNA were, respectively, transfected into VSMCs for 72 hours, and the knockdown efficiency was detected. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the NC group.) (e) Nile Red (orange) was used to label the lipid, and the concentration of intracellular lipid was measured. ( n = 5 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the untreated group; @@ P < 0.01, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ## P < 0.01, the oxLDL-treated PP2 group compared with the oxLDL-treated PP3 group.) (f, g) Western blot was conducted to measure the VSMC contractile phenotype markers (Myh11 and α Sma) and foam cell markers (Mac2 and Cd68). All the western blot results were calculated using grayscale value, and β -actin served as an internal reference gene to normalize protein expression. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the control group (untreated group); @@ P < 0.01, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ## P < 0.01, the oxLDL-treated PP2 group compared with oxLDL-treated PP3 group).

Article Snippet: Additionally, primary antibodies of β -actin (Cat#3700), p-Src Y418 (Cat#6943), t-Src (Cat#2123), Mnsod (Cat#13141), Sirt1 (Cat#2314), Sirt2 (Cat#12650), Sirt3 (Cat#5490), Sirt5 (Cat#8782), Sirt6 (Cat#12486), Sirt7 (Cat#5360), and tlr4 (Cat#14358) as well as the second antibody-conjunction HRP anti-mouse/rabbit (Cat#7076/Cat#7074) were gained from CST (MA, USA).

Techniques: Activation Assay, Western Blot, Transfection, Blocking Assay, Negative Control, Concentration Assay, Expressing

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