arp2 3 ab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc arp2 3 ab
    Primers used for RT-PCR.
    Arp2 3 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    arp2 3 ab - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair"

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    Journal: Stem Cells International

    doi: 10.1155/2019/1513526

    Primers used for RT-PCR.
    Figure Legend Snippet: Primers used for RT-PCR.

    Techniques Used: Sequencing

    Reagents used for in vitro experiments.
    Figure Legend Snippet: Reagents used for in vitro experiments.

    Techniques Used: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

    JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.
    Figure Legend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Techniques Used: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

    arp2 3 ab  (Cell Signaling Technology Inc)


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  • 94

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    Cell Signaling Technology Inc arp2 3 ab
    Primers used for RT-PCR.
    Arp2 3 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/arp2 3 ab/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    arp2 3 ab - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair"

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    Journal: Stem Cells International

    doi: 10.1155/2019/1513526

    Primers used for RT-PCR.
    Figure Legend Snippet: Primers used for RT-PCR.

    Techniques Used: Sequencing

    Reagents used for in vitro experiments.
    Figure Legend Snippet: Reagents used for in vitro experiments.

    Techniques Used: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

    JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.
    Figure Legend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Techniques Used: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

    anti arp2 3  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
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    Cell Signaling Technology Inc anti arp2 3
    Primers used for RT-PCR.
    Anti Arp2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti arp2 3/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti arp2 3 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair"

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    Journal: Stem Cells International

    doi: 10.1155/2019/1513526

    Primers used for RT-PCR.
    Figure Legend Snippet: Primers used for RT-PCR.

    Techniques Used: Sequencing

    Reagents used for in vitro experiments.
    Figure Legend Snippet: Reagents used for in vitro experiments.

    Techniques Used: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

    JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.
    Figure Legend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Techniques Used: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

    rabbit polyclonal antibodies against arp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal antibodies against arp2
    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified <t>polyclonal</t> antibodies against the <t>Arp2/3</t> complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
    Rabbit Polyclonal Antibodies Against Arp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal antibodies against arp2/product/Cell Signaling Technology Inc
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    rabbit polyclonal antibodies against arp2 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction"

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008478

    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
    Figure Legend Snippet: Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

    Techniques Used: Cell Culture, Labeling, Binding Assay, Affinity Purification

    Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.
    Figure Legend Snippet: Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

    Techniques Used: Activation Assay

    anti arp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti arp2
    Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, <t>ARP2,</t> and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.
    Anti Arp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti arp2/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti arp2 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVβ3 and Mediating αVβ3/Src/E2F1 Signaling"

    Article Title: Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVβ3 and Mediating αVβ3/Src/E2F1 Signaling

    Journal: Journal of Cancer

    doi: 10.7150/jca.26740

    Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, ARP2, and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.
    Figure Legend Snippet: Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, ARP2, and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.

    Techniques Used: Inverted Microscopy, Western Blot

    anti arp 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti arp 2
    Anti Arp 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti arp 2/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    anti arp 2 - by Bioz Stars, 2023-02
    94/100 stars

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    ceacam1 d3r80  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ceacam1 d3r80
    Ceacam1 D3r80, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    actr2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc actr2
    (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the <t>ACTR2</t> KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).
    Actr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    1) Product Images from "Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages"

    Article Title: Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1010184

    (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).
    Figure Legend Snippet: (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).

    Techniques Used: Microscopy, Infection, Western Blot, Stable Transfection, Expressing, CRISPR

    anti arp2  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc anti arp2
    a The protein expression analysis of actin polymerization positive (red) and negative (green) regulators by western blot in MV4-1/MOLM-13 MID-Res compared to parental cells, in MV4-11/MOLM-13 MID-Res ± 50 nM Mid compared to MID-Sens ± 50 nM Mid. b In FLT3 KD MV4-11/MOLM-13 MID-Res compared to scramble MV4-11 MID-Res and in MV4-11 MID-Res treated with Mid and Eht alone/combination. GAPDH was used as housekeeping. c Proliferation and Mid IC50 (after 4 days) in MV4-11/MOLM-13 MID-Res N-WASP KD ((MV) p < 0.0001, t = 25.7, df = 3.8, (MO) ( p < 0.001, t = 10.5, df = 3.9)), WAVE2 KD ((MV) p = 0.32, t = 1.1, df = 2.93, (MO) p = 0.4, t = 1, df = 2.6), <t>ARP2/3</t> complex KD ((MV) p < 0.001, t = 39.6, df = 2.06, (MO) p < 0.001, t = 11.8, df = 3.6), and PFN1 KD ((MV) p < 0.001, t = 25.1, df = 3.9, (MO) p < 0.001, t = 9.8, df = 3.5) compared to scramble MID-Res cells. d Actin polymerization positive regulators protein expression analysis targeting them by siRNAs in MV4-11/MOLM-13 MID-Res. e Correlation analysis between the mean of actin polymerization positive regulators (N-WASP, WAVE2, ARP2, PFN1) and FLT3 pathway genes expression in 639 AML patients by using microarray expression data (E-MTAB-3444). The western blot results are normalized by loading control (GAPDH) and are expressed as fold change relative to the control. Data are shown as means ± SDs (error bars) from three independent experiments.
    Anti Arp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia"

    Article Title: Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02215-w

    a The protein expression analysis of actin polymerization positive (red) and negative (green) regulators by western blot in MV4-1/MOLM-13 MID-Res compared to parental cells, in MV4-11/MOLM-13 MID-Res ± 50 nM Mid compared to MID-Sens ± 50 nM Mid. b In FLT3 KD MV4-11/MOLM-13 MID-Res compared to scramble MV4-11 MID-Res and in MV4-11 MID-Res treated with Mid and Eht alone/combination. GAPDH was used as housekeeping. c Proliferation and Mid IC50 (after 4 days) in MV4-11/MOLM-13 MID-Res N-WASP KD ((MV) p < 0.0001, t = 25.7, df = 3.8, (MO) ( p < 0.001, t = 10.5, df = 3.9)), WAVE2 KD ((MV) p = 0.32, t = 1.1, df = 2.93, (MO) p = 0.4, t = 1, df = 2.6), ARP2/3 complex KD ((MV) p < 0.001, t = 39.6, df = 2.06, (MO) p < 0.001, t = 11.8, df = 3.6), and PFN1 KD ((MV) p < 0.001, t = 25.1, df = 3.9, (MO) p < 0.001, t = 9.8, df = 3.5) compared to scramble MID-Res cells. d Actin polymerization positive regulators protein expression analysis targeting them by siRNAs in MV4-11/MOLM-13 MID-Res. e Correlation analysis between the mean of actin polymerization positive regulators (N-WASP, WAVE2, ARP2, PFN1) and FLT3 pathway genes expression in 639 AML patients by using microarray expression data (E-MTAB-3444). The western blot results are normalized by loading control (GAPDH) and are expressed as fold change relative to the control. Data are shown as means ± SDs (error bars) from three independent experiments.
    Figure Legend Snippet: a The protein expression analysis of actin polymerization positive (red) and negative (green) regulators by western blot in MV4-1/MOLM-13 MID-Res compared to parental cells, in MV4-11/MOLM-13 MID-Res ± 50 nM Mid compared to MID-Sens ± 50 nM Mid. b In FLT3 KD MV4-11/MOLM-13 MID-Res compared to scramble MV4-11 MID-Res and in MV4-11 MID-Res treated with Mid and Eht alone/combination. GAPDH was used as housekeeping. c Proliferation and Mid IC50 (after 4 days) in MV4-11/MOLM-13 MID-Res N-WASP KD ((MV) p < 0.0001, t = 25.7, df = 3.8, (MO) ( p < 0.001, t = 10.5, df = 3.9)), WAVE2 KD ((MV) p = 0.32, t = 1.1, df = 2.93, (MO) p = 0.4, t = 1, df = 2.6), ARP2/3 complex KD ((MV) p < 0.001, t = 39.6, df = 2.06, (MO) p < 0.001, t = 11.8, df = 3.6), and PFN1 KD ((MV) p < 0.001, t = 25.1, df = 3.9, (MO) p < 0.001, t = 9.8, df = 3.5) compared to scramble MID-Res cells. d Actin polymerization positive regulators protein expression analysis targeting them by siRNAs in MV4-11/MOLM-13 MID-Res. e Correlation analysis between the mean of actin polymerization positive regulators (N-WASP, WAVE2, ARP2, PFN1) and FLT3 pathway genes expression in 639 AML patients by using microarray expression data (E-MTAB-3444). The western blot results are normalized by loading control (GAPDH) and are expressed as fold change relative to the control. Data are shown as means ± SDs (error bars) from three independent experiments.

    Techniques Used: Expressing, Western Blot, Microarray

    anti arp2 3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti arp2 3
    Schematic presentation of the cell shape and stress fibre network in static and laminar shear stress or LXN deficiency ECs. In normal condition, transverse arcs are generated from <t>Arp2/3,</t> α‐actinin and FLNA crosslinked actin filaments in normal ECs. Under laminar shear stress, LXN loss resulted in the proteolytic cleavage and subcellular localization of FLNA, down‐regulation of Arp2/3, α‐actinin and paxillin in ECs. As a result, ECs were elongated from the typical cobblestone pattern to uniformly fusiform and showed the distribution of F‐actin filaments in longitudinal direction of the cell
    Anti Arp2 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "LXN deficiency regulates cytoskeleton remodelling by promoting proteolytic cleavage of Filamin A in vascular endothelial cells"

    Article Title: LXN deficiency regulates cytoskeleton remodelling by promoting proteolytic cleavage of Filamin A in vascular endothelial cells

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.16685

    Schematic presentation of the cell shape and stress fibre network in static and laminar shear stress or LXN deficiency ECs. In normal condition, transverse arcs are generated from Arp2/3, α‐actinin and FLNA crosslinked actin filaments in normal ECs. Under laminar shear stress, LXN loss resulted in the proteolytic cleavage and subcellular localization of FLNA, down‐regulation of Arp2/3, α‐actinin and paxillin in ECs. As a result, ECs were elongated from the typical cobblestone pattern to uniformly fusiform and showed the distribution of F‐actin filaments in longitudinal direction of the cell
    Figure Legend Snippet: Schematic presentation of the cell shape and stress fibre network in static and laminar shear stress or LXN deficiency ECs. In normal condition, transverse arcs are generated from Arp2/3, α‐actinin and FLNA crosslinked actin filaments in normal ECs. Under laminar shear stress, LXN loss resulted in the proteolytic cleavage and subcellular localization of FLNA, down‐regulation of Arp2/3, α‐actinin and paxillin in ECs. As a result, ECs were elongated from the typical cobblestone pattern to uniformly fusiform and showed the distribution of F‐actin filaments in longitudinal direction of the cell

    Techniques Used: Generated

    antibodies against arp2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibodies against arp2
    A and B , Representative immunofluorescence photographs of <t>Arp2</t> ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.
    Antibodies Against Arp2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization"

    Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.121.020870

    A and B , Representative immunofluorescence photographs of Arp2 ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.
    Figure Legend Snippet: A and B , Representative immunofluorescence photographs of Arp2 ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.

    Techniques Used: Immunofluorescence, Knock-Out, Fluorescence, Derivative Assay, Western Blot, Transfection, Small Interfering RNA, Expressing

    A through C , A10 VSMCs were treated with either a low (10 ng/mL) or high (1000 ng/mL) concentration of recombinant MFG‐E8 for 16 hours. A , Western blot analysis of the protein expression of Arp2 and actin‐related protein (Arp3) in A10 cells treated with low and high doses of MFG‐E8 for 16 hours. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) levels normalized to those of α‐tubulin were conducted (n=3). Data are presented as mean±SD. Three independent experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D through E , VSMCs isolated from aortas of wild‐type mice were immunostained with antibodies against Arp2 ( D ) and Arp3 ( E ) and costained with phalloidin to label filamentous actin (F‐actin). Bar, 50 μm. Three independent experiments were performed and each experiment was repeated with similar results. In a representative experiment, the fluorescence intensities of Arp2 (n=3–5) ( D ) and Arp3 (n=3) ( E ) were quantitated. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. * P <0.05 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. DAPI indicates 4',6‐diamidino‐2‐phenylindole.
    Figure Legend Snippet: A through C , A10 VSMCs were treated with either a low (10 ng/mL) or high (1000 ng/mL) concentration of recombinant MFG‐E8 for 16 hours. A , Western blot analysis of the protein expression of Arp2 and actin‐related protein (Arp3) in A10 cells treated with low and high doses of MFG‐E8 for 16 hours. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) levels normalized to those of α‐tubulin were conducted (n=3). Data are presented as mean±SD. Three independent experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D through E , VSMCs isolated from aortas of wild‐type mice were immunostained with antibodies against Arp2 ( D ) and Arp3 ( E ) and costained with phalloidin to label filamentous actin (F‐actin). Bar, 50 μm. Three independent experiments were performed and each experiment was repeated with similar results. In a representative experiment, the fluorescence intensities of Arp2 (n=3–5) ( D ) and Arp3 (n=3) ( E ) were quantitated. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. * P <0.05 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

    Techniques Used: Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Isolation, Fluorescence

    A through C , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. A , Immunoblotting was performed to assess the protein expression of Arp2 and actin‐related protein 3 (Arp3) in A10 cells in a representative experiment. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) normalized to GAPDH were conducted (n=3). Data are presented as mean±SD. Three repeated experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D , Representative confocal fluorescent images of phalloidin for filamentous actin (F‐actin) (green) and DNase I for globular actin (G‐actin) (red) staining of A10 VSMCs stimulated with low (10 ng/mL) and high (1000 ng/mL) doses of MFG‐E8 for 16 hours. Bar, 50 μm. In a representative experiment, corresponding quantification of the mean fluorescence intensity of phalloidin for F‐actin (n=5) and that of DNase I for G‐actin (n=4–6) was performed. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. ** P <0.01 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.
    Figure Legend Snippet: A through C , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. A , Immunoblotting was performed to assess the protein expression of Arp2 and actin‐related protein 3 (Arp3) in A10 cells in a representative experiment. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) normalized to GAPDH were conducted (n=3). Data are presented as mean±SD. Three repeated experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D , Representative confocal fluorescent images of phalloidin for filamentous actin (F‐actin) (green) and DNase I for globular actin (G‐actin) (red) staining of A10 VSMCs stimulated with low (10 ng/mL) and high (1000 ng/mL) doses of MFG‐E8 for 16 hours. Bar, 50 μm. In a representative experiment, corresponding quantification of the mean fluorescence intensity of phalloidin for F‐actin (n=5) and that of DNase I for G‐actin (n=4–6) was performed. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. ** P <0.01 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

    Techniques Used: Incubation, Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Staining, Fluorescence

    A , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. Rac1 activity was measured using the G‐LISA (Cytoskeleton) assay. Data represent the averages of duplicate samples (n=2), and the error bars indicate the SD. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. B and C , A10 cells were incubated with the Rac1 inhibitor NSC23766 (200 μM, Tocris) 16 hours before treatment with low and high doses of MFG‐E8 for 16 hours. B , Immunoblotting analysis of the Arp2 protein expression in A10 VSMCs was performed. C , Quantitative analysis of Arp2 normalized to GAPDH was performed (n=4). Data are presented as mean±SD. Four repeated experiments were performed. Each point is derived from each of the 4 repeated experiments. * P <0.05 and ** P <0.01, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.
    Figure Legend Snippet: A , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. Rac1 activity was measured using the G‐LISA (Cytoskeleton) assay. Data represent the averages of duplicate samples (n=2), and the error bars indicate the SD. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. B and C , A10 cells were incubated with the Rac1 inhibitor NSC23766 (200 μM, Tocris) 16 hours before treatment with low and high doses of MFG‐E8 for 16 hours. B , Immunoblotting analysis of the Arp2 protein expression in A10 VSMCs was performed. C , Quantitative analysis of Arp2 normalized to GAPDH was performed (n=4). Data are presented as mean±SD. Four repeated experiments were performed. Each point is derived from each of the 4 repeated experiments. * P <0.05 and ** P <0.01, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

    Techniques Used: Incubation, Concentration Assay, Recombinant, Activity Assay, Western Blot, Expressing, Derivative Assay

    Common carotid arteries (CCAs) of wild‐type (WT) mice were ligated, and recombinant MFG‐E8 (2 μg/mL) was delivered using pluronic gel into the vessels. A and B , Representative immunofluorescence images display the expression of Arp2 ( A ) and Arp3 ( B ) in the sham‐operated CCAs, ligated CCAs, and ligated CCAs treated with high‐dose MFG‐E8 in WT mice 21 days after ligation. Lumen (L), neointima (I), and media (M) of the vessels are indicated. Bar, 50 μm. C , Protein levels of Arp2 were quantified through immunoblotting. Proteins were extracted from 10 pooled CCAs per group for Western blotting and examined using antibodies against Arp2. In the quantitative analysis, the Arp2 and actin‐related protein 3 (Arp3) levels were normalized to that of β‐tubulin. DAPI indicates 4',6‐diamidino‐2‐phenylindole.
    Figure Legend Snippet: Common carotid arteries (CCAs) of wild‐type (WT) mice were ligated, and recombinant MFG‐E8 (2 μg/mL) was delivered using pluronic gel into the vessels. A and B , Representative immunofluorescence images display the expression of Arp2 ( A ) and Arp3 ( B ) in the sham‐operated CCAs, ligated CCAs, and ligated CCAs treated with high‐dose MFG‐E8 in WT mice 21 days after ligation. Lumen (L), neointima (I), and media (M) of the vessels are indicated. Bar, 50 μm. C , Protein levels of Arp2 were quantified through immunoblotting. Proteins were extracted from 10 pooled CCAs per group for Western blotting and examined using antibodies against Arp2. In the quantitative analysis, the Arp2 and actin‐related protein 3 (Arp3) levels were normalized to that of β‐tubulin. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

    Techniques Used: Recombinant, Immunofluorescence, Expressing, Ligation, Western Blot

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    Cell Signaling Technology Inc arp2 3 ab
    Primers used for RT-PCR.
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    Primers used for RT-PCR.
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    Cell Signaling Technology Inc rabbit polyclonal antibodies against arp2
    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified <t>polyclonal</t> antibodies against the <t>Arp2/3</t> complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).
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    Cell Signaling Technology Inc anti arp2
    Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, <t>ARP2,</t> and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.
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    Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, <t>ARP2,</t> and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.
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    Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, <t>ARP2,</t> and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.
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    Cell Signaling Technology Inc actr2
    (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the <t>ACTR2</t> KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).
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    Cell Signaling Technology Inc antibodies against arp2
    A and B , Representative immunofluorescence photographs of <t>Arp2</t> ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.
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    Image Search Results


    Primers used for RT-PCR.

    Journal: Stem Cells International

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    doi: 10.1155/2019/1513526

    Figure Lengend Snippet: Primers used for RT-PCR.

    Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

    Techniques: Sequencing

    Reagents used for in vitro experiments.

    Journal: Stem Cells International

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    doi: 10.1155/2019/1513526

    Figure Lengend Snippet: Reagents used for in vitro experiments.

    Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

    Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

    JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Journal: Stem Cells International

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    doi: 10.1155/2019/1513526

    Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Article Snippet: Arp2/3 Ab , Western blot , 0.1 μ g/ml , 24 h , CST, USA.

    Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

    Primers used for RT-PCR.

    Journal: Stem Cells International

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    doi: 10.1155/2019/1513526

    Figure Lengend Snippet: Primers used for RT-PCR.

    Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

    Techniques: Sequencing

    Reagents used for in vitro experiments.

    Journal: Stem Cells International

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    doi: 10.1155/2019/1513526

    Figure Lengend Snippet: Reagents used for in vitro experiments.

    Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

    Techniques: In Vitro, Concentration Assay, FACS, Flow Cytometry, Immunofluorescence, Western Blot

    JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Journal: Stem Cells International

    Article Title: Bone Marrow-Derived CD44 + Cells Migrate to Tissue-Engineered Constructs via SDF-1/CXCR4-JNK Pathway and Aid Bone Repair

    doi: 10.1155/2019/1513526

    Figure Lengend Snippet: JNK served as an effector downstream of SDF-1/CXCR4 in the migration of bone marrow (BM) CD44 + cells towards tissue-engineered constructs (TECs). (a) Comparison of phosphorylated JNK (p-JNK), ERK (p-ERK), and P38 (p-P38) expression in BM cells after CXCR4 blockade. After migration stopped, BM cells were collected and analyzed by western blot. (b) Representative images of cell migration towards SDF-1. The quantification of migrated BM cells is shown as a bar graph ( n = 5). ∗∗ P < 0.01. (c) FACS analysis of the proportions of CD44 + cells in peripheral blood (PB). The quantification comparison is shown as a bar graph ( n = 3). ∗∗ P < 0.01. (d) Representative images of in vivo migration of GFP + /CD44 + cells towards TECs. The introduction of SP600125 significantly reduced the recruitment of GFP + /CD44 + cells. White triangle, implant area; white arrows, CD44 + cells; scale bar, 50 mm. (e) Comparison of Arpin and Arp2/3 expressions in BM cells after JNK blockade in vitro . (f) Analysis of the Arpin, Arp2, and Arp3 mRNA expression in sorted CD44 + cells. On day 3 postoperatively, cells in PB were sorted by FACS on CD44. RT-PCR was performed to evaluate Arpin, Arp2, and Arp3 mRNA expression. The quantification data are shown as a bar graph ( n = 3). ∗∗ P < 0.01.

    Article Snippet: After blocking with 5% milk, membranes were incubated overnight at 4°C with the following primary antibodies: anti-CXCR4, anti-p-ERK, anti-p38, anti-p-JNK, and anti-Arpin (1 : 1000 dilution; Abcam, USA), and anti-Arp2/3 (1 : 1000 dilution; CST, USA), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000 dilution; SouthernBiotech, Birmingham, AL) at room temperature for 1 hour.

    Techniques: Migration, Construct, Expressing, Western Blot, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction

    Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Cultured embryonic Xenopus muscle cells labeled with rhodamine-α-bungarotoxin (R-BTX) were stimulated overnight with polystyrene beads coated with heparan-binding growth-associated molecule (HB-GAM) (A, D; asterisks) to induce AChR clusters (C, F). Cells were then fixed and labeled with affinity-purified polyclonal antibodies against the Arp2/3 complex proteins Arp2 (B) and p34arc (E) followed by FITC-linked anti-rabbit secondary antibodies. Separately, bead-stimulated muscle cells (G) were labeled with anti-p34arc polyclonal (H) and anti-cortactin monoclonal (I; mAb4F11) antibodies and then FITC-conjugated anti-rabbit and rhodamine-conjugated anti-mouse secondary antibodies. AChRs, Arp2 and p34arc were clustered at bead-muscle contacts (A-F; arrows) where cortactin localized and overlapped in distribution with p34arc (H and I; arrows). In primary muscle cultures non-muscle cells were occasionally found (J) and in these cells p34arc (K) and cortactin (L) localized along the cell periphery (arrowheads) but were not clustered at bead-cell contacts (“b” in K and L corresponds to bead indicated by asterisk in J).

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Cell Culture, Labeling, Binding Assay, Affinity Purification

    Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

    Journal: PLoS ONE

    Article Title: The Function of Cortactin in the Clustering of Acetylcholine Receptors at the Vertebrate Neuromuscular Junction

    doi: 10.1371/journal.pone.0008478

    Figure Lengend Snippet: Activation of MuSK by agrin induces AChR clustering in an actin polymerization-dependent manner. This model depicts a possible way in which cortactin signaling might promote the AChR clustering process. Initiation of intracellular signaling by the activated MuSK complex could enhance cortactin's tyrosine phosphorylation through src family tyrosine kinases (SFKs) (and possibly other kinases such as abl), and cortactin, in turn, could increase actin polymerization. Alternatively, cortactin might trigger actin polymerization by activating the Arp2/3 complex, either on its own or in concert with WASP-related proteins (N-WASP, WIP, etc.) to which it could be linked by the adapter Nck. In parallel, via other signaling intermediates, MuSK could stimulate Rho-family GTPases and, through them, F-actin assembly. Such enhanced and dynamic actin polymerization at synaptic sites could generate a scaffold which “traps” AChRs through rapsyn.

    Article Snippet: These reagents were purchased: rhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR, USA); monoclonal antibodies against cortactin (4F11) and phosphotyrosine (4G10) and a rabbit polyclonal antibody against p34arc (Upstate Biotechnology; Lake Placid, NY, USA); rabbit polyclonal antibodies against cortactin phosphorylated on Y421, Y466 or Y482 (Cell Signaling Technology; Danvers, MA, USA); rabbit polyclonal antibodies against Arp2 and Y390-phosphorylated AChR β-subunit (Santa Cruz Biotechnology; Santa Cruz, CA, USA); monoclonal anti-Shp2 and anti-neurexin-1 antibodies (BD Biosciences; San Jose, CA, USA); monoclonal anti-α-tubulin antibody DM1A (Sigma; St Louis, MO, USA); rhodamine- and FITC-conjugated secondary antibodies (Zymed; South San Francisco, CA, USA); horseradish-peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research Laboratories; West Grove, PA, USA); and Triton X-100 (TX-100) and West Pico enhanced chemiluminescence (ECL) reagent (Pierce; Rockford, IL, USA).

    Techniques: Activation Assay

    Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, ARP2, and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.

    Journal: Journal of Cancer

    Article Title: Antimetastatic Effect of Fucoidan-Sargassum against Liver Cancer Cell Invadopodia Formation via Targeting Integrin αVβ3 and Mediating αVβ3/Src/E2F1 Signaling

    doi: 10.7150/jca.26740

    Figure Lengend Snippet: Fucoidan-Sargassum inhibits the initiation and assembly stages of invadopodia formation in HCCLM3 cells. (A) The elongated protrusions of the HCCLM3 cells, which are called invadopodia; the image was taken under an inverted microscope (400× magnification). (B) Western blotting analysis of FAK, Src, and Cortactin, which are key proteins involved in the initiation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h. (C) Confocal images of fixed control or Fucoidan-Sargassum-treated cells immunostained for actin (2nd panel), cortactin (middle panel), and Src (4th panel). The arrowheads indicate active invadopodia initiation in the control cells and the cytoskeletal changes in the treated cells. Scale bars: 7.5 µm. (D and E) Western blotting analysis of TKS5, N-WASP, ARP3, ARP2, and CDC42, which are key proteins involved in the assembly stage of invadopodia formation, and MMP2 and MT1-MMP, which are involved in the maturation stage of invadopodia formation, after the cells were treated with Fucoidan-Sargassum (0, 10, 20, and 30 mg/mL) for 48 h.

    Article Snippet: Rabbit polyclonal antibodies were used: anti-Tublin, anti-IRE1a were from Beyotime Biotechnology (Shanghai, China), anti-TKS5, anti-ITGα1 were from Zen-Biotechnology (Sichuan, China), anti-GRP78, aniti-GRP94, anti-SPARC, anti-c-Src were from Proteintech (Rosemont, IL, USA), anti-ITGαV, anti-ITGβ3, anti-ITGβ1, anti-Src, anti-phospho-Src416, anti-phospho-cortactin, anti-FAK, anti-phospho-FAK, anti-ARP2, anti-ARP3, anti-N-WASP, anti-WAVE-2, anti-Rac1/Cdc42, anti-phospho-Rac1/Cdc42(ser71), anti-E2F1, anti-phospho -E2F1, anti-phospho-MPZL1 were from Cell Signaling Technology (Danvers, MA, USA), anti-MPZL was from Abcam (Cambridge, MA, USA).

    Techniques: Inverted Microscopy, Western Blot

    (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).

    Journal: PLoS Pathogens

    Article Title: Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

    doi: 10.1371/journal.ppat.1010184

    Figure Lengend Snippet: (A-E) Gonococci internalization by U937 MFs (A-D, H and J) and hMDMs (E) requires formin-mediated actin polymerization but not canonical phagocytosis. (A-E, H and J) Quantitative analysis of bacteria uptake by macrophages using inside/out microscopy in the presence/absence of different inhibitors added 30 min prior to infection. Gonococcal infections were stopped at 8hpi and the percentage of intracellular colonies in each condition was calculated (green bars). L . pneumophila ΔdotA infections were stopped at 2hpi and the percentage of internalized bacteria was determined (gray bars). The internalization index reflects how each treatment affects uptake compared to vehicle control and was calculated for each condition by dividing the percentage of internalized objects from the treatment condition by the percentage of internalized objects from the respective vehicle control condition, which was then multiplied by 100. Inhibitors targeting: (A) actin polymerization (cytochalasin D, 5μM) and microtubules polymerization (nocodazole, 3μM); (B) the small GTPases Cdc42 (ML-141, 5μM), Rac1/2/3 (EHT 1864, 5μM) and the downstream effectors of Rho GTPase—LIMK1/2 kinases (BMS-5, 1μM) and ROCK1/2 kinases (GSK269962, 1μM); (C) the phagosome regulators Dynamin (dynasore, 30μM) and Phosphoinositide-3 kinase (LY294002, 10μM); (D) actin polymerization regulators formins (SMIFH2, 12.5 or 25μM), Arp2/3 complex (CK869, 10μM), Myosin II ATPases ((-) blebbistatin, from 2.5 to 20μM). (E) Decreased gonococci uptake at 8hpi by hMDMs treated with SMIFH2 as indicated. (F) Immunoblot analysis of the U937 cell line stably expressing Cas9 used for CRISPR genome editing. (G) Immunoblot validation for the loss of Arp2 in the ACTR2 KO and LC3 in the MAP1LC3B KO U937 MFs. (H and J) N . g colony internalization is not affected by the loss of (H) Arp2 or (J) LC3. (I) mRNA expression for the indicated genes in U937 cells from RNAseq presented in normalized transcript per million (nTPMs). Data obtained from the Human Atlas Project : MAP1LC3A ( https://www.proteinatlas.org/ENSG00000101460-MAP1LC3A/cell+line ); MAP1LC3B ( https://www.proteinatlas.org/ENSG00000140941-MAP1LC3B/cell+line ); MAP1LC3C ( https://www.proteinatlas.org/ENSG00000197769-MAP1LC3C/cell+line ). (A-E, H and J) Means from at least three biological replicates ± SD. At least 100 N . g colonies or L . p ΔdotA bacteria for each condition were scored. The statistical significance of the differences between the internalization index from inhibitor-treated cells and the vehicle-treated cells for each condition were calculated using the unpaired T-test (A-C, D for Arp2/3INH, H and J) or one-way ANOVA (D for Formin INH and MyosinII INH , E) , not significant (ns.).

    Article Snippet: Rabbit monoclonal antibodies against the following antigens were purchased from Cell Signaling—Rab5 (C8B1), Rab7 (D95F2), EEA1 (C45B10), GM130 (D6B1), Ezrin (#3145), LC3A/B (D3U4C), V5-Tag (E9H80), CEACAM1 (D3R80) and ACTR2 (#3128S).

    Techniques: Microscopy, Infection, Western Blot, Stable Transfection, Expressing, CRISPR

    A and B , Representative immunofluorescence photographs of Arp2 ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

    doi: 10.1161/JAHA.121.020870

    Figure Lengend Snippet: A and B , Representative immunofluorescence photographs of Arp2 ( A ) and actin‐related protein 3 (Arp3) ( B ) in the carotid arteries of wild‐type (WT) and MFG‐E8 knockout (KO) mice. Bar, 50 μm. Quantification of fluorescence intensities of Arp2 (WT: n mice =5, MFG‐E8 knockout: n mice =5) and Arp3 (WT: n mice =7, MFG‐E8 knockout: n mice =7) is shown in ( A ) and ( B ), respectively. Results are presented as mean±SEM. Each point is derived from an assessment of 3 sections of an individual animal. **** P <0.0001, as obtained using the t test. C , Proteins were extracted from 10 pooled common carotid arteries of WT and knockout mice for Western blotting and examined with antibodies against Arp2 and Arp3. In the quantitative analysis, the Arp2 and Arp3 levels were normalized to that of GAPDH. D , A10 VSMCs underwent 48‐hour transfection with small interfering RNA (siRNA) against MFG‐E8 (si‐MFG‐E8) or control siRNA (Ctrl siRNA). The expression of Arp2 and Arp3 was evaluated through Western blotting. The Arp2 (n=3) and Arp3 (n=3) levels were normalized to those of α‐tubulin. Three repeated experiments were performed. Data are presented as mean±SD. Each point is derived from each of the 3 repeated experiments. ** P <0.01, as obtained using the t test. CCA indicates common carotid artery; and DAPI, 4',6‐diamidino‐2‐phenylindole.

    Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

    Techniques: Immunofluorescence, Knock-Out, Fluorescence, Derivative Assay, Western Blot, Transfection, Small Interfering RNA, Expressing

    A through C , A10 VSMCs were treated with either a low (10 ng/mL) or high (1000 ng/mL) concentration of recombinant MFG‐E8 for 16 hours. A , Western blot analysis of the protein expression of Arp2 and actin‐related protein (Arp3) in A10 cells treated with low and high doses of MFG‐E8 for 16 hours. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) levels normalized to those of α‐tubulin were conducted (n=3). Data are presented as mean±SD. Three independent experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D through E , VSMCs isolated from aortas of wild‐type mice were immunostained with antibodies against Arp2 ( D ) and Arp3 ( E ) and costained with phalloidin to label filamentous actin (F‐actin). Bar, 50 μm. Three independent experiments were performed and each experiment was repeated with similar results. In a representative experiment, the fluorescence intensities of Arp2 (n=3–5) ( D ) and Arp3 (n=3) ( E ) were quantitated. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. * P <0.05 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

    doi: 10.1161/JAHA.121.020870

    Figure Lengend Snippet: A through C , A10 VSMCs were treated with either a low (10 ng/mL) or high (1000 ng/mL) concentration of recombinant MFG‐E8 for 16 hours. A , Western blot analysis of the protein expression of Arp2 and actin‐related protein (Arp3) in A10 cells treated with low and high doses of MFG‐E8 for 16 hours. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) levels normalized to those of α‐tubulin were conducted (n=3). Data are presented as mean±SD. Three independent experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D through E , VSMCs isolated from aortas of wild‐type mice were immunostained with antibodies against Arp2 ( D ) and Arp3 ( E ) and costained with phalloidin to label filamentous actin (F‐actin). Bar, 50 μm. Three independent experiments were performed and each experiment was repeated with similar results. In a representative experiment, the fluorescence intensities of Arp2 (n=3–5) ( D ) and Arp3 (n=3) ( E ) were quantitated. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. * P <0.05 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

    Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

    Techniques: Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Isolation, Fluorescence

    A through C , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. A , Immunoblotting was performed to assess the protein expression of Arp2 and actin‐related protein 3 (Arp3) in A10 cells in a representative experiment. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) normalized to GAPDH were conducted (n=3). Data are presented as mean±SD. Three repeated experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D , Representative confocal fluorescent images of phalloidin for filamentous actin (F‐actin) (green) and DNase I for globular actin (G‐actin) (red) staining of A10 VSMCs stimulated with low (10 ng/mL) and high (1000 ng/mL) doses of MFG‐E8 for 16 hours. Bar, 50 μm. In a representative experiment, corresponding quantification of the mean fluorescence intensity of phalloidin for F‐actin (n=5) and that of DNase I for G‐actin (n=4–6) was performed. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. ** P <0.01 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

    doi: 10.1161/JAHA.121.020870

    Figure Lengend Snippet: A through C , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. A , Immunoblotting was performed to assess the protein expression of Arp2 and actin‐related protein 3 (Arp3) in A10 cells in a representative experiment. Quantitative analyses of Arp2 ( B ) and Arp3 ( C ) normalized to GAPDH were conducted (n=3). Data are presented as mean±SD. Three repeated experiments were performed. Each point is derived from each of the 3 repeated experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. D , Representative confocal fluorescent images of phalloidin for filamentous actin (F‐actin) (green) and DNase I for globular actin (G‐actin) (red) staining of A10 VSMCs stimulated with low (10 ng/mL) and high (1000 ng/mL) doses of MFG‐E8 for 16 hours. Bar, 50 μm. In a representative experiment, corresponding quantification of the mean fluorescence intensity of phalloidin for F‐actin (n=5) and that of DNase I for G‐actin (n=4–6) was performed. Data are presented as mean±SD. Each point is derived from an assessment of each picture field. ** P <0.01 and **** P <0.0001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

    Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

    Techniques: Incubation, Concentration Assay, Recombinant, Western Blot, Expressing, Derivative Assay, Staining, Fluorescence

    A , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. Rac1 activity was measured using the G‐LISA (Cytoskeleton) assay. Data represent the averages of duplicate samples (n=2), and the error bars indicate the SD. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. B and C , A10 cells were incubated with the Rac1 inhibitor NSC23766 (200 μM, Tocris) 16 hours before treatment with low and high doses of MFG‐E8 for 16 hours. B , Immunoblotting analysis of the Arp2 protein expression in A10 VSMCs was performed. C , Quantitative analysis of Arp2 normalized to GAPDH was performed (n=4). Data are presented as mean±SD. Four repeated experiments were performed. Each point is derived from each of the 4 repeated experiments. * P <0.05 and ** P <0.01, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

    doi: 10.1161/JAHA.121.020870

    Figure Lengend Snippet: A , A10 vascular smooth muscle cells (VSMCs) incubated with either 50 μg/mL β1 inhibitory antibody (Itgb1 Ab) or isotype control 4 hours before treatment with either a low or high concentration of recombinant MFG‐E8 for 16 hours. Rac1 activity was measured using the G‐LISA (Cytoskeleton) assay. Data represent the averages of duplicate samples (n=2), and the error bars indicate the SD. * P <0.05, ** P <0.01, *** P <0.001, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test. B and C , A10 cells were incubated with the Rac1 inhibitor NSC23766 (200 μM, Tocris) 16 hours before treatment with low and high doses of MFG‐E8 for 16 hours. B , Immunoblotting analysis of the Arp2 protein expression in A10 VSMCs was performed. C , Quantitative analysis of Arp2 normalized to GAPDH was performed (n=4). Data are presented as mean±SD. Four repeated experiments were performed. Each point is derived from each of the 4 repeated experiments. * P <0.05 and ** P <0.01, as obtained using 1‐way ANOVA followed by Tukey multiple comparisons test.

    Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

    Techniques: Incubation, Concentration Assay, Recombinant, Activity Assay, Western Blot, Expressing, Derivative Assay

    Common carotid arteries (CCAs) of wild‐type (WT) mice were ligated, and recombinant MFG‐E8 (2 μg/mL) was delivered using pluronic gel into the vessels. A and B , Representative immunofluorescence images display the expression of Arp2 ( A ) and Arp3 ( B ) in the sham‐operated CCAs, ligated CCAs, and ligated CCAs treated with high‐dose MFG‐E8 in WT mice 21 days after ligation. Lumen (L), neointima (I), and media (M) of the vessels are indicated. Bar, 50 μm. C , Protein levels of Arp2 were quantified through immunoblotting. Proteins were extracted from 10 pooled CCAs per group for Western blotting and examined using antibodies against Arp2. In the quantitative analysis, the Arp2 and actin‐related protein 3 (Arp3) levels were normalized to that of β‐tubulin. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: MFG‐E8 Regulates Vascular Smooth Muscle Cell Migration Through Dose‐Dependent Mediation of Actin Polymerization

    doi: 10.1161/JAHA.121.020870

    Figure Lengend Snippet: Common carotid arteries (CCAs) of wild‐type (WT) mice were ligated, and recombinant MFG‐E8 (2 μg/mL) was delivered using pluronic gel into the vessels. A and B , Representative immunofluorescence images display the expression of Arp2 ( A ) and Arp3 ( B ) in the sham‐operated CCAs, ligated CCAs, and ligated CCAs treated with high‐dose MFG‐E8 in WT mice 21 days after ligation. Lumen (L), neointima (I), and media (M) of the vessels are indicated. Bar, 50 μm. C , Protein levels of Arp2 were quantified through immunoblotting. Proteins were extracted from 10 pooled CCAs per group for Western blotting and examined using antibodies against Arp2. In the quantitative analysis, the Arp2 and actin‐related protein 3 (Arp3) levels were normalized to that of β‐tubulin. DAPI indicates 4',6‐diamidino‐2‐phenylindole.

    Article Snippet: To determine the expression of Arp2 and Arp3 in the mouse CCAs, the paraffinized sections within the first millimeter proximal to the ligature were deparaffinized, permeabilized with 0.1% Triton‐X 100 in PBS for 10 minutes, blocked with 5% normal goat serum in 0.1% Triton‐X 100 in PBS, and incubated with primary antibodies against Arp2 and Arp3 (1:200, Cell Signaling) overnight at 4°C; this was followed by an additional incubation with Alexa Fluor 594–conjugated secondary antibodies (1:100, Thermo Fisher Invitrogen) for 1 hour at room temperature.

    Techniques: Recombinant, Immunofluorescence, Expressing, Ligation, Western Blot