Journal: BMC Cell Biology

Article Title: Inhibition of cytokinesis by wiskostatin does not rely on N-WASP/Arp2/3 complex pathway

doi: 10.1186/1471-2121-9-42

Figure Lengend Snippet: Arp3 and/or N-WASP knockdowns do not affect cytokinesis . A. HeLa cells were transfected with water (Ø) or specific siRNA directed against N-WASP or Arp3 or N-WASP + Arp3 or ECT2. 72 hours after transfection cells were harvested and lysed. N-WASP, Arp3, ECT2, p34 and β-tubulin protein levels were determined by immunoblot analysis using specific antibodies. B. The DNA content of the same HeLa cells was determined by flow cytometry analysis. Arrows indicate abnormal DNA content in ECT2 knockdown cells. A similar scale was used for each histogram. These results are representative of at least three independent experiments. C. Representative DIC images from time-lapse movies of HeLa cells transfected with water (Ø), siRNA directed against N-WASP (N-WASP), against Arp3 (Arp3), against Arp3 and N-WASP (Arp3 + N-WASP) or against ECT2. Images started to be acquired 24 hours post-transfection for 40 hours. Coloured stars indicate the nuclei of dividing cells and of their daughter cells. Time indicated as hours: minutes.

Article Snippet: Polyclonal antibody against p34 (ARPC2) was from Upstate Biotechnology.

Techniques: Transfection, Western Blot, Flow Cytometry, Knockdown

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: The Journal of Neuroscience

Article Title: The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse

doi: 10.1523/JNEUROSCI.1321-16.2016

Figure Lengend Snippet: The Arp2/3 actin polymerization complex is downregulated after BLOC-1 loss-of-function. A–C, SH-SY5Y cells were treated with control or shRNAs against either one of three BLOC-1 subunits (Bloc1s5, 6, and 8) and cell extracts analyzed by immunoblot with antibodies against the BLOC-1-sensitive proteome components: Arp2/3 complex subunits (Arpc2 and 5), ataxin 2 (Atxn2), and plexin A2 (PlxnA2). Antibodies against actin, BLOC-1 subunits, and the BLOC-1-sensitive SNARE VAMP7 were used as controls. D, MNT1 melanoma cells were treated with control or Bloc1s6 shRNAs and cell extracts analyzed as in A. E, Immortalized melanocytes from mouse mutants carrying null mutations in the BLOC-1 subunits Bloc1s5–6 (Bloc1s5mu/mu and Bloc1s6pa/pa) or cells rescued by expression of the missing subunit were analyzed as in A. F, G, Synaptosomes of six wild-type and six Bloc1s8sdy/sdy hippocampi were analyzed. F, G, Three independent fractionations. In F, all fractions are depicted (H, homogenate and P1, low speed pellet), whereas in G, only synaptosomes are shown. Samples were probed by immunoblot with Arpc2 and 5 antibodies, hsp90 as loading control, dysbindin, and the synaptic vesicle marker SV to indicate synapse enrichment. A1–G1, Dot plots representing the protein expression normalized to the control genotype. Each dot depicts an independent determination from at least three independent experiments. Red line marks 100%. All quantitations in A1 to E1 were significantly different from actin. In F1 and G1, only Arpc5 was significantly different compared with controls with hsp90 (F, G). Significance was determined by Kruskal–Wallis test followed by Wilcoxon Mann–Whitney nonparametric tests.

Article Snippet: Animal procedures and studies were approved by the Emory University Institutional Animal Care and Use Committee. . Antibodies against rabbit ArpC2 and ArpC5 (15058-1-AP and 16717-1-AP, 1:1000 blot dilution; ProteinTech Group), rabbit ArpC2 used for immunoprecipitation (07-227, 1 μg; Millipore), rabbit plexin A2 (6896, 1:1000 blot dilution; Cell Signaling Technology), rabbit dysbindin (HPA029616, 1:125 blot dilution; Sigma-Aldrich), rabbit ataxin 2 (A301-118A, 1:2000 blot dilution; Bethyl), mouse VAMP7 (1:750 blot dilution; a gift from Dr. A.A. Peden, Sheffield University, UK), mouse pallidin clone 2G6 (1:500 blot dilution; a gift from Dr. Esteban Dell'Angelica, University of California–Los Angeles), rabbit FAM21 (MC2188, 1:2000 blot dilution; a gift from Dr. Daniel Billadeau, Mayo Cilinic, Rochester, MN), rabbit strumpellin (SC87442, 1:1000 blot dilution; Santa Cruz Biotechnology), mouse actin clone AC-15 (A5451, 1:500 blot dilution; Sigma-Aldrich) mouse monoclonal SV2 (Clone 10H, 1:500 blot dilution; Developmental Studies Hybridoma Bank), mouse monoclonal HSP90 (610418, blot dilution 1:1000; BD Biosciences), and mouse monoclonal FLAG (M2 clone, F-3165, 1 μg used for immunoprecipitation, blot dilution 1:1000; Sigma-Aldrich). .

Techniques: Control, Western Blot, Expressing, Marker, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse

doi: 10.1523/JNEUROSCI.1321-16.2016

Figure Lengend Snippet: Genetic and biochemical interactions of the BLOC-1, WASH, and Arp2/3 complexes. A, Control (WASHf/f) and WASH-null (WASH−/−) mouse embryonic fibroblast cell extracts were analyzed by immunoblot with antibodies against the BLOC-1-sensitive proteome components Arp2/3 complex subunits (Arpc2 and 5) and ataxin 2 (Atxn2), as well as antibodies against actin, BLOC-1 subunits (Bloc1s6 and 8), and the WASH complex subunits strumpellin and FAM21. B, Dot plots representing the protein expression normalized to the control genotype. Each dot represents an independent determination from three or four independent experiments. Red line marks 100%. p-values were obtained by comparing against actin using the Kruskal–Wallis test followed by Wilcoxon Mann–Whitney nonparametric tests. Arpc2 and BLOC-1 subunits were not significant (NS). Asterisks mark all significant values (p < 0.0017). D, E, Immunoprecipitations of FLAG-tagged dysbindin expressed in SH-SY5Y cells (D) or immunoprecipitations with Arpc2 antibodies (E). BLOC-1 complex was detected by immunoblot of the precipitated complexes with antibodies against FLAG or pallidin, WASH complexes were revealed by blotting with antibodies against strumpellin and FAM21; the Arp2/3 complex was detected with antibodies against Arpc2 (D, E, n = 3).

Article Snippet: Animal procedures and studies were approved by the Emory University Institutional Animal Care and Use Committee. . Antibodies against rabbit ArpC2 and ArpC5 (15058-1-AP and 16717-1-AP, 1:1000 blot dilution; ProteinTech Group), rabbit ArpC2 used for immunoprecipitation (07-227, 1 μg; Millipore), rabbit plexin A2 (6896, 1:1000 blot dilution; Cell Signaling Technology), rabbit dysbindin (HPA029616, 1:125 blot dilution; Sigma-Aldrich), rabbit ataxin 2 (A301-118A, 1:2000 blot dilution; Bethyl), mouse VAMP7 (1:750 blot dilution; a gift from Dr. A.A. Peden, Sheffield University, UK), mouse pallidin clone 2G6 (1:500 blot dilution; a gift from Dr. Esteban Dell'Angelica, University of California–Los Angeles), rabbit FAM21 (MC2188, 1:2000 blot dilution; a gift from Dr. Daniel Billadeau, Mayo Cilinic, Rochester, MN), rabbit strumpellin (SC87442, 1:1000 blot dilution; Santa Cruz Biotechnology), mouse actin clone AC-15 (A5451, 1:500 blot dilution; Sigma-Aldrich) mouse monoclonal SV2 (Clone 10H, 1:500 blot dilution; Developmental Studies Hybridoma Bank), mouse monoclonal HSP90 (610418, blot dilution 1:1000; BD Biosciences), and mouse monoclonal FLAG (M2 clone, F-3165, 1 μg used for immunoprecipitation, blot dilution 1:1000; Sigma-Aldrich). .

Techniques: Control, Western Blot, Expressing, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Changes in E-cadherin rigidity sensing regulate cell adhesion

doi: 10.1073/pnas.1618676114

Figure Lengend Snippet: Validation of small molecule inhibitors. (A) MDCK cells adhered to Ecad-Fc substrates and stained for the formin protein FMN1 in the presence or absence of the small molecule inhibitor SMIFH2. (Scale bar, 10 µm.) (B) MDCK cells adhered to Ecad-Fc substrates and stained for the Arp2/3 subunit ARPC2 in the presence or absence of the Arp 2/3 inhibitor CK666. (Scale bar, 10 µm.) (C) GTP-bound Cdc42 isolated from control and ML141-treated cells and analyzed by SDS/PAGE. Bar graph indicates the average from three independent experiments. Activity levels were normalized by setting the control condition to “1.” Error bars represent SEM. (D) GTP-bound Rac isolated from control and NSC23766-treated cells and analyzed by SDS/PAGE. Bar graph indicates the average from three independent experiments. Activity levels were normalized by setting the control condition to 1. Error bars represent SEM.

Article Snippet: The following primary antibodies were used: Arp2 (anti-p34-Arc/ARPC2; Millipore), formin1 (anti-FMN1; Novus Biologicals), and paxillin (anti-paxillin; BD Biosciences).

Techniques: Biomarker Discovery, Staining, Isolation, Control, SDS Page, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Changes in E-cadherin rigidity sensing regulate cell adhesion

doi: 10.1073/pnas.1618676114

Figure Lengend Snippet: Cell adhesion to a 30-kPa and 60-kPa Ecad-Fc PA gel requires the activities of distinct signaling molecules. (A–D) Representative montage of MDCK cells stably expressing E-cadherin:dsRed adhered to a 30-kPa or 60-kPa Ecad-Fc PA gel. Each panel is representative of the effects on cell protrusive area following the addition and washout of different inhibitors: (A) pan-formin inhibitor, SMIFH2; (B) Arp2/3 inhibitor, CK666; (C) Cdc42 inhibitor, ML141; and (D) Rac inhibitor, NSC23766. (Scale bar, 10 µm.) (A–D, Right) Line graphs represent the mean protrusive area for each condition throughout the time course of the experiment. For each line graph, the mean protrusive area of cells on a 30-kPa Ecad-Fc PA gel and on a 60-kPa Ecad-Fc PA gel is indicated by the green and blue lines, respectively. The gray region between the dotted lines indicates the period in which the inhibitor was present. n ≥ 10 cells per condition from at least three independent experiments; error bars represent SEM.

Article Snippet: The following primary antibodies were used: Arp2 (anti-p34-Arc/ARPC2; Millipore), formin1 (anti-FMN1; Novus Biologicals), and paxillin (anti-paxillin; BD Biosciences).

Techniques: Stable Transfection, Expressing