Journal: International Journal of Molecular Sciences

Article Title: Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

doi: 10.3390/ijms141020220

Figure Lengend Snippet: List of VSMC proteins up or downregulated by H 2 O 2 treatment in a Nox1-dependent manner.

Article Snippet: Seventy two hours after transfection, VSMC lysates were subjected to SDS-PAGE/Western blot with a polyclonal antibody against ARPC2 (Aviva Systems Biology, San Diego, CA, USA), total p38 MAPK (Cell Signaling) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and monoclonal antibody against phospho-p38 MAPK (Cell Signaling).

Techniques: Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

doi: 10.3390/ijms141020220

Figure Lengend Snippet: Upregulation of ARPC2 protein expression in VSMCs via Nox1. Nox1 and Scrmb siRNA-treated VSMC were incubated with vehicle or 50 μM H 2 O 2 for 3 h. VSMC lysates were subjected to Western blot and probed with a polyclonal antibody against ARPC2 (Aviva Systems Biology) and β-actin (Santa Cruz Biotechnology). ( A ) Representative Western blot; ( B ) Bar graphs representing averaged optical density data expressed as a ratio of ARPC2 to β-actin ( n = 6). Data represent the mean ± SEM. * p < 0.05 vs . Scrmb siRNA + vehicle treatment; # p < 0.05 vs . Scrmb siRNA + H 2 O 2 treatment.

Article Snippet: Seventy two hours after transfection, VSMC lysates were subjected to SDS-PAGE/Western blot with a polyclonal antibody against ARPC2 (Aviva Systems Biology, San Diego, CA, USA), total p38 MAPK (Cell Signaling) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and monoclonal antibody against phospho-p38 MAPK (Cell Signaling).

Techniques: Expressing, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

doi: 10.3390/ijms141020220

Figure Lengend Snippet: H 2 O 2 stimulates VSMC migration via ARPC2. ARPC2 and Scrmb siRNA-transfected VSMC were incubated with vehicle or H 2 O 2 (50 μM) for 24 h. Cell migration was determined by wounding of VSMC monolayers. Images were captured at 0 and 24 h. ( A ) Representative Western blot showing ARPC2 and β-actin expression in Scrmb- and ARPC2-siRNA-treated VSMC. Images are representative of three independent experiments with similar results; ( B ) Representative images of wound healing at 0 and 24 h after scratch are shown (original magnification, ×5). Dashed lines denote the approximate edge of the wound at 0 and 24 h time points; ( C ) Quantitative assessment of VSMC migration ( n = 6). Data represent the mean ± SEM. * p < 0.05 vs . Scrmb siRNA + vehicle treatment; # p < 0.05 vs . Scrmb siRNA + H 2 O 2 treatment.

Article Snippet: Seventy two hours after transfection, VSMC lysates were subjected to SDS-PAGE/Western blot with a polyclonal antibody against ARPC2 (Aviva Systems Biology, San Diego, CA, USA), total p38 MAPK (Cell Signaling) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and monoclonal antibody against phospho-p38 MAPK (Cell Signaling).

Techniques: Migration, Transfection, Incubation, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Analysis Identifies an NADPH Oxidase 1 (Nox1)-Mediated Role for Actin-Related Protein 2/3 Complex Subunit 2 (ARPC2) in Promoting Smooth Muscle Cell Migration

doi: 10.3390/ijms141020220

Figure Lengend Snippet: Inhibition of p38 MAPK pathway attenuates H 2 O 2 -induced ARPC2 expression. VSMC were pre-treated with the p38 MAPK inhibitor (p38i) SB203580 (1 μM, 1 h, Millipore), followed by treatment with vehicle or H 2 O 2 (50 μM) for 3 h. VSMC lysates were subjected to Western blot and probed with polyclonal antibodies against ARPC2 and β-actin. ( A ) Representative Western blot showing ARPC2 and β-actin expression in control- and p38i-treated VSMC incubated with vehicle or H 2 O 2 ; ( B ) Bar graphs representing averaged optical density data expressed as a ratio of ARPC2 to β-actin ( n = 4). Data represent the mean ± SEM. * p < 0.05 vs . Control + vehicle treatment. # p < 0.05 vs . Control + H 2 O 2 treatment.

Article Snippet: Seventy two hours after transfection, VSMC lysates were subjected to SDS-PAGE/Western blot with a polyclonal antibody against ARPC2 (Aviva Systems Biology, San Diego, CA, USA), total p38 MAPK (Cell Signaling) and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) and monoclonal antibody against phospho-p38 MAPK (Cell Signaling).

Techniques: Inhibition, Expressing, Western Blot, Control, Incubation

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Several subunits of the Arp2/3 and CCT complexes were identified in the PAK4 interactome. White nodes: proteins passed QMS cut-off; Grey nodes: proteins appeared in MS but did not pass the QMS cut-off; Darker grey node: not in the MS list. (B) After GFP-Trap IP of H1299 cell lysates transiently expressing EGFP (control) or EGFP-PAK4, samples were subjected to immunoblot analysis for the indicated proteins. The upper panel is blotted with anti-CCTε, the middle panel with anti-ARPC2, while anti-GFP was used to control the IP efficiency in the lower panel. Input lanes are direct immunoblot of the used cell lysates. (C) After anti-CCTε IP of H1299 cell lysates transiently expressing EGFP-PAK4, blots were probed with an anti-GFP antibody in the upper panel. Anti-CCTε was used to control the IP efficiency in the lower panel. Input lane shows direct immunoblotting of the used lysate.

Article Snippet: 1000 μg protein lysate were immunoprecipated by GFP-Trap (gta-20, ChromoTek), CCTε and N-WASP antibodies with rabbit IgG as control, separated and probed with rat CCTε antibody (MCA2178, BIO-RAD), rabbit ARPC2 (HPA008352, Atlas Antibodies) and rabbit N-WASP antibody (HPA005750, Atlas Antibodies) and mouse GFP antibody (MAB2510, Milipore).

Techniques: Expressing, Control, Western Blot

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet: ( A ) dSTORM reconstruction of F-actin stained with phalloidin-AlexaFluor647 in synapses of primary mouse B cells interacting for 20 min with PMS loaded with anti-Igκ F(ab’) 2 . The cells were treated with the indicated inhibitors from 5 min after initial spreading. Scale bar, 2 μm. ( B ) Quantification of foci characteristics obtained from TIRF timelapses of live cells treated with inhibitors as in ( A ). Plots show foci numbers, mean lifetimes, and mean displacements per cell. ( C ) Immunoblot of lysates of Cas9 Ramos cells transfected with the indicated gRNAs, developed with anti-ARPC2 and anti-GAPDH antibodies. ( D, F ) TIRF images of F-actin stained with phalloidin-AlexaFluor647 in synapses of a Cas9-expressing Ramos B cells transduced with the indicated gRNAs. Cells were imaged after interacting for 20 min with PMS loaded with anti-IgM F(ab’) 2 . Scale bars, 5 μm. ( E, G ) Quantification of Ramos actin foci numbers per cell area. Data in B, E, G show values from individual cells pooled from two ( B, G ) or three ( E ) experiments. Bars indicate means and SDs. P, significance in one-way ANOVA compared to DMSO or control, *, p<0.0001.

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Staining, Western Blot, Transfection, Expressing, Transduction, Control

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet: ( A ) Ramos cells expressing ARPC2-mRuby (magenta) were imaged by TIRF microscopy on PMSs loaded with anti-IgM F(ab’) 2 . F-actin was stained with phalloidin-AlexaFluor647 (green). Scale bar, 5 μm. Panels on the right show magnified area in the white box. Arrows show ARPC2 clusters colocalized with actin foci. Scale bar 1 μm. ( B ) Example of dynamics of ARPC2-mRuby in a single actin focus visualized with Lifeact-GFP. Time zero corresponds to initial focus formation. Scalebar 1 μm. ( C ) Example of a dynamic filament growth from ARPC2-positive actin foci in Ramos cells co-expressing ARPC2-mRuby and Lifeact-GFP. Bottom panel shows results of actin and fiber segmentation. Scalebar 1 μm. ( D ) Ramos cells expressing DIAPH1-mRuby (magenta) were imaged as in ( A ). Scale bar, 5 μm. Panels on the right show magnified area in white box. Arrows show clusters of DIAPH1 colocalized with actin foci. Scale bars 1 μm. ( E ) Example of dynamics of DIAPH1-mRuby in a single actin focus visualized with Lifeact-GFP. Time zero corresponds to initial focus formation. Scalebar 1 μm. ( F ) Example of a fiber outgrow from a DIAPH1 cluster in . Scalebar 1 μm. ( G ) Quantification of relative enrichment or depletion of ARPC2-mRuby and DIAPH1-mRuby fluorescence in actin foci and filaments. Data are mean ± SEM from n = 4 experiments each containing 12–213 cells. P, significance from one-way ANOVA. ( H, I ) Fraction of actin foci colocalized with ARPC2 ( G ) or DIAPH1 ( H ) spots and vice versa. Total bar heights show rates of colocalization, white bars indicate colocalization obtained after randomization of locations of the corresponding structures. Values are mean ± SEM of n = 4 of the same experiments as in F. P, significance in two-way repeated measures ANOVA comparing the measured with the randomized data.

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Expressing, Microscopy, Staining, Fluorescence

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet: TIRF images of a Ramos cell expressing ARPC2-mRuby and Lifeact-GFP, and labeled with the plasma membrane dye DiD on PMS loaded with anti-IgM F(ab’) 2 . Magnified inset shows an ARPC2-negative actin fiber associated with an ARPC2-positive actin focus in the absence of DiD membrane signal (arrow), in contrast to DiD-labeled filopodia at the cell periphery (arrowheads). Scalebar 5 μm (1 μm in inset). Contrast was enhanced in inset images.

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Expressing, Labeling, Clinical Proteomics, Membrane

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet:

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Concentration Assay, Staining

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet:

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: