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: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Basic characterization of the G-quadruplex motifs under investigation. ( A ) and ( B ) CD spectra and melting curves of the G-quadruplex motifs and their mutated control oligonucleotides (A: MMP16; B: ARPC2). ( C ) Melting temperature at different concentrations of the ARPC2 G-quadruplex. All melting experiments were carried out at 100 mM KCl, except for the MMP16 G-quadruplex-forming sequence, since it did not unfold in the measured temperature range. D) RT-PCR experiments to confirm expression of MMP16 and ARPC2 in HEK293 cells. Additional experiments were carried out with HeLa cells. Control experiments without reverse transcription are shown (–).

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Circular Dichroism, Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Reverse Transcription

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Inhibition of translation by the G-quadruplex motifs of MMP16 and ARPC2, as determined by dual luciferase assays. ( A ) Dual luciferase assays for the isolated G-quadruplex motifs and ( B ) for the full-length 5′-UTR of ARPC2. The ratio of Renilla and firefly luciferase activity is normalized to the value of the empty psiCHECK-2 vector. The G-quadruplex motif in the 5′-UTR of Renilla luciferase reduces enzyme synthesis. Values given are averages ± standard deviations of three experiments.

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Inhibition, Luciferase, Isolation, Activity Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Pull-down assays. ( A ) and ( B ) G-quadruplex motifs of the MMP16 (A) and ARPC2 (B) mRNAs were coupled to streptavidin agarose beads. Whole-cell extract of HEK293 cells was added to the beads. After several washing steps, proteins were eluted with buffers containing the indicated K + -concentrations. Proteins were analyzed by SDS-PAGE. Proteins that bind exclusively to the G-quadruplex-sequence (Q) are indicated. GQ-M represents an independent experiment, in which the HEK293 extract was first incubated with the mutated oligonucleotide and then with the G-quadruplex-forming RNA. The indicated proteins were identified by MS-based peptide mass fingerprinting. ( C ) Pull-down assay with the additional ARPC2 control oligonucleotide with two G-stretches (ARPC2–2xG). 1. ME2 (66 kDa); 2. YB-1 (36 kDa); 3. U2AF65 (54 kDa); 4. hnRNPH (50 kDa); 5. YB-1 (36 kDa) hnRNPF (46 kDa); 6. RPS2 (32 kDa); 7. Nucl (76 kDa); 8. RBM14 (70 kDa); 9. SRSF1 (28 kDa); 10. SRSF1 (28 kDa) RPS6 (29 kDa); 11. RPL7 (29 kDa); 12. SRSF9 (26 kDa), RPS6 (29 kDa); 13. SRSF9 (26 kDa), RPL14 (23,5 kDa); 14. RPL10 (25 kDa) RPS9 (23 kDa); 15. RPL26 (17 kDa); 16. RPL27a (17 kDa); 17. RPS9 (23 kDa); a. Actin (42 kDa); b. hnRNPA3 (39 kDa); c. hnRNPA2B1 (37 kDa) and hnRNPA3 (39 kDa); d. hnRNPA2B1 (37 kDa); e. hnRNPA1 (39 kDa); f. YB-1 (36 kDA); g. YB-1/Actin (36 kDa, 42 kDa).

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: SDS Page, Sequencing, Incubation, Peptide Mass Fingerprinting, Pull Down Assay, Control

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Proteins identified to bind to the G-quadruplex motifs of the ARPC2 and MMP16 mRNA

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Protein Binding, RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Association ( k a ) and dissociation ( k d ) rates and dissociation constants (K D ) determined by SPR spectroscopy; n. d.: not determined due to too weak interactions

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Spectroscopy

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Analysis of protein-RNA interactions by SPR spectroscopy. ( A ) Binding parameters for Δnucleolin to the ARPC2 and MMP16 G-quadruplex and the mutated sequence were analyzed in the concentration range of 1–20 nM and 10–1000 nM, respectively. Data were fitted to 1:1 binding with mass transfer. Sensorgrams are shown in gray and the respective fits in black. ( B ) Binding of U2AF65 to 100 nM of the ARPC2 and MMP16 G-quadruplex oligonucleotides and experiments with the respective controls. ( C ) Additional experiments with the G-rich control oligonucleotides ARPC2 2xG in comparison to the G-quadruplex and the exhaustively mutated oligonucleotide.

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Spectroscopy, Binding Assay, Sequencing, Concentration Assay, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Analysis of protein-RNA interactions by SPR spectroscopy. Binding of SRSF1 ( A ) and EFHD2 ( B ) to 100 nM of the G-quadruplex and mutated control sequences of ARPC2 and MMP16.

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Spectroscopy, Binding Assay, Control