Journal: Scientific Reports

Article Title: A conformational change within the WAVE2 complex regulates its degradation following cellular activation

doi: 10.1038/srep44863

Figure Lengend Snippet: ( a ) Jurkat T cells expressing YFP-WAVE2 were left unstimulated (−), or were co-stimulated with anti-CD3 and anti-CD28 antibodies (+). MG132-treated cells were also left unstimulated (−) or co-stimulated (+, MG132). An equal number of cells were lysed for each sample. Cell lysates were immunoprecipitated using anti-GFP antibody and immunoblotted for ubiquitin. Similar amounts of WAVE2 were precipitated for each sample by using the precipitating antibody as the limiting factor. Ubiquitylated YFP-WAVE2 appears as a smear of bands around 110 kDa. Data shown are representative of four independent experiments. ( b ) Jurkat T cells were left unstimulated (−), or were co-stimulated with anti-CD3 and anti-CD28 antibodies (+). Cell lysates were immunoprecipitated using anti-Ub antibody and immunoblotted for WAVE2. Ubiquitylated endogenous WAVE2 appears as a smear of bands above the MW of 75 kDa with a prominent band around 83 kDa. Data shown are representative of three independent experiments. ( c ) Upper panel: Human primary peripheral-blood lymphocytes (PBLs) were left unstimulated (−) or were co-stimulated with anti-CD3 and anti-CD28 antibodies (+). MG132-treated cells were also left unstimulated (−) or co-stimulated (+, MG132). An equal number of cells were lysed for each sample. Cell lysates were immunoprecipitated using anti-WAVE2 antibody and immunoblotted for ubiquitin. Similar amounts of WAVE2 were precipitated for each sample by using the precipitating antibody as the limiting factor. Ubiquitylated endogenous WAVE2 appears as a smear of bands above the MW of 75 kDa, with a prominent band at ~83 kDa. Data shown are representative of at least three independent experiments. Lower panel: Cell lysates were prepared from activated and non-activated PBLs that were either pretreated with MG132 or left untreated, and analyzed for WAVE2 protein levels. WAVE2 expression levels were measured by ImageJ. Densitometry results for WAVE2 after normalization relative to the GAPDH values are presented.

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: mouse anti-GFP, rat anti-HA peroxidase 3F10 (Roche Applied Science); mouse anti-GAPDH (Biodesign); rabbit anti-ubiquitin (Dako); rabbit anti-Hem-1 (Novus); goat anti-WAVE2 D16, mouse anti-ubiquitin P4D1, mouse anti-Vav D7 (Santa Cruz Biotechnology, Inc); rabbit anti-phospho WAVE2 (Ser-351), mouse anti-phosphotyrosine (pTy) 4G10 (Millipore).

Techniques: Expressing, Immunoprecipitation, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: A conformational change within the WAVE2 complex regulates its degradation following cellular activation

doi: 10.1038/srep44863

Figure Lengend Snippet: ( a (i)) Schemes of YFP-WAVE2 wt and YFP-WAVE2 deletion mutants. WHD , WAVE homology domain; B , basic region; PRD , proline-rich domain; VCA , verprolin-homology cofilin-homology acidic domain. ( a( ii)) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms as indicated. YFP-WAVE2 was immunoprecipitated using anti-GFP antibody; complexes were resolved by SDS-PAGE and immunoblotted for ubiquitin using anti-HA antibody, and for YFP-WAVE2 using anti-GFP antibody. Ubiquitylated YFP-WAVE2 wt appears at a MW of ~110 kDa, which is 8 kDa above the MW of YFP-WAVE2 (~102 kDa). Data shown are representative of at least three independent experiments. ( b ) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms as indicated. Cells were lysed and immunoprecipitated with anti-GFP. IPs were separated by SDS-PAGE, transferred, and blotted with anti-HA and anti-GFP antibodies. Data shown are representative of three independent experiments. ( c (i)) Schematic illustration of WAVE2 structural domains. The WAVE2 WHD domain contains ten lysine residues, as indicated. ( c (ii)) Schemes of YFP-WAVE2 deletion mutants. ( c (iii)) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms described in cii. Cells were lysed and immunoprecipitated with anti-GFP. IPs were separated by SDS-PAGE, transferred, and blotted with anti-HA and anti-GFP antibodies. Data shown are representative of at least four independent experiments. ( d ) 293 T cells were co-transfected, immunoprecipitated, and blotted as described above. Densitometric analysis of the bands presented was performed using ImageJ and normalized to the GFP densitometry values. Error bars show standard error of the mean (SEM). Significance was determined by two-tailed Student’s t-test and is indicated by asterisks (* P < 0.05). Data shown are representative of at least three independent experiments.

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: mouse anti-GFP, rat anti-HA peroxidase 3F10 (Roche Applied Science); mouse anti-GAPDH (Biodesign); rabbit anti-ubiquitin (Dako); rabbit anti-Hem-1 (Novus); goat anti-WAVE2 D16, mouse anti-ubiquitin P4D1, mouse anti-Vav D7 (Santa Cruz Biotechnology, Inc); rabbit anti-phospho WAVE2 (Ser-351), mouse anti-phosphotyrosine (pTy) 4G10 (Millipore).

Techniques: Transfection, Construct, Ubiquitin Proteomics, Mutagenesis, Immunoprecipitation, SDS Page, Two Tailed Test

Journal: Scientific Reports

Article Title: A conformational change within the WAVE2 complex regulates its degradation following cellular activation

doi: 10.1038/srep44863

Figure Lengend Snippet: ( a ) Jurkat T cells were transfected with siRNA specific to human Hem-1 as well as with a non-specific (N.S) scrambled siRNA control. After 24 h, cells were treated with MG132 or left untreated, followed by co-stimulation with anti-CD3 and anti-CD28 antibodies. Cell lysates were prepared and analyzed for Hem-1 and WAVE2 protein levels. The gene-silencing efficiencies of Hem-1 as well as WAVE2 expression levels were measured by ImageJ. Densitometry results for Hem-1 or WAVE2 after normalization relative to the GAPDH values are presented. Vertical lines indicate noncontiguous blots. All lanes are from the same film and exposure time. ( b ) Lysates described in ( a ) from cells that were not treated with MG132, were immunoprecipitated using anti-WAVE2 antibody and immunoblotted for ubiquitin. Ubiquitylated endogenous WAVE2 appears above the MW of 75 kDa with a prominent band at ~83 kDa. ( c ) Jurkat T cells expressing YFP-WAVE2 wt or the YFP-WAVE2 mutant forms, Δ25-64aa and K45R, were sorted for YFP-positive cells. Cells were co-stimulated with anti-CD3 and anti-CD28 antibodies followed by lysis. Lysates were analyzed for WAVE2 protein levels by immunoblotting with anti-GFP antibody or anti-GAPDH as a loading control. Densitometric analysis of the bands presented was performed using ImageJ and normalized to the GAPDH densitometry values. Data shown are representative of three independent experiments.

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: mouse anti-GFP, rat anti-HA peroxidase 3F10 (Roche Applied Science); mouse anti-GAPDH (Biodesign); rabbit anti-ubiquitin (Dako); rabbit anti-Hem-1 (Novus); goat anti-WAVE2 D16, mouse anti-ubiquitin P4D1, mouse anti-Vav D7 (Santa Cruz Biotechnology, Inc); rabbit anti-phospho WAVE2 (Ser-351), mouse anti-phosphotyrosine (pTy) 4G10 (Millipore).

Techniques: Transfection, Control, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Mutagenesis, Lysis, Western Blot

Journal: Scientific Reports

Article Title: A conformational change within the WAVE2 complex regulates its degradation following cellular activation

doi: 10.1038/srep44863

Figure Lengend Snippet: ( a (i)) Schemes of YFP-WAVE2 deletion mutants. ( a (ii)) 293 T cells were transfected with constructs encoding YFP-WAVE2 wt or YFP-WAVE2 mutant forms as indicated. Cells were lysed and analyzed for WAVE2 protein levels by immunoblotting with anti-GFP antibody or anti-GAPDH as a loading control. Densitometric analysis of the bands presented was performed using ImageJ and normalized to the GAPDH densitometry values. Data shown are representative of (blot), or averages of (lower graph) five independent experiments. Error bars show SEM. Significance was determined by two-tailed Student’s t-test and is indicated by asterisks (* P < 0.05; ** P < 0.005; *** P < 0.0005). ( b ) 293 T cells were co-transfected with constructs encoding HA-tagged ubiquitin together with YFP-WAVE2 wt or with YFP-WAVE2 mutant forms, as indicated. YFP-WAVE2 was immunoprecipitated by anti-GFP, and the membranes were blotted with anti-HA and anti-GFP. Ubiquitylated YFP-WAVE2 wt appears at a MW of ~110 kDa. Densitometric analysis of the bands presented was performed using ImageJ and normalized to the GFP densitometry values. Data shown are representative of (blot), or averages of (lower graph) three independent experiments. Error bars show SEM. Significance was determined by two-tailed Student’s t-test, and is indicated by asterisks (* P < 0.05; ** P < 0.005; *** P < 0.0005). ( c (i)) Scheme of 81-183aa-deleted YFP-WAVE2. ( c (ii)) 293 T cells were transfected with constructs encoding YFP-WAVE2 wt or YFP-WAVE2 mutant forms, as indicated. Cells were left untreated or pretreated with 0.5 μM MG132 overnight, lysed, and analyzed by immunoblotting, as described above. Densitometric analysis of the bands was performed using ImageJ and normalized to the GAPDH densitometry values.

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: mouse anti-GFP, rat anti-HA peroxidase 3F10 (Roche Applied Science); mouse anti-GAPDH (Biodesign); rabbit anti-ubiquitin (Dako); rabbit anti-Hem-1 (Novus); goat anti-WAVE2 D16, mouse anti-ubiquitin P4D1, mouse anti-Vav D7 (Santa Cruz Biotechnology, Inc); rabbit anti-phospho WAVE2 (Ser-351), mouse anti-phosphotyrosine (pTy) 4G10 (Millipore).

Techniques: Transfection, Construct, Mutagenesis, Western Blot, Control, Two Tailed Test, Ubiquitin Proteomics, Immunoprecipitation

Journal: Scientific Reports

Article Title: A conformational change within the WAVE2 complex regulates its degradation following cellular activation

doi: 10.1038/srep44863

Figure Lengend Snippet: ( a (i)) Schematic representation of the tagged WAVE2 protein used for FRET analysis. ( a (ii)) Jurkat cells expressing YFP-WAVE2-CFP and untransfected cells (negative control – Neg. Cont.) were analyzed by western blotting with anti-GFP. GAPDH served as a loading control. ( a (iii)) Jurkat T cells expressing YFP-WAVE2-CFP were plated over either non-stimulatory coverslips coated with anti-CD43 antibody (upper panel, n = 89 cells), or over stimulatory coverslips coated with anti-CD3 antibody (lower panel, n = 42 cells). Cells were fixed following 3 min of activation and imaged by confocal microscopy. FRET analysis was performed using the donor-sensitized acceptor emission technology (see Methods for details). A graph summarizing the mean FRET efficiency in cells expressing YFP-WAVE2-CFP plated over the indicated coverslips is presented on the right. Graph shows means ± SEM from three independent experiments. Significance was determined by two-tailed Student’s t-test. ( b (i)) Scheme of 81-183aa-deleted YFP-WAVE2-CFP. ( b (ii)) Jurkat T cells transfected with a plasmid encoding YFP-WAVE2-CFP Δ81-183aa were plated over either non-stimulatory (anti-CD43, upper panel, n = 76 cells) or stimulatory (anti-CD3, lower panel, n = 48 cells) coverslips and fixed after 3 min of activation. A graph showing the mean FRET efficiency in cells expressing YFP-WAVE2-CFP Δ81-183aa plated over the indicated coverslips is presented on the right. Graph shows means ± SEM from three independent experiments. Significance was determined by two-tailed Student’s t-test.

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: mouse anti-GFP, rat anti-HA peroxidase 3F10 (Roche Applied Science); mouse anti-GAPDH (Biodesign); rabbit anti-ubiquitin (Dako); rabbit anti-Hem-1 (Novus); goat anti-WAVE2 D16, mouse anti-ubiquitin P4D1, mouse anti-Vav D7 (Santa Cruz Biotechnology, Inc); rabbit anti-phospho WAVE2 (Ser-351), mouse anti-phosphotyrosine (pTy) 4G10 (Millipore).

Techniques: Expressing, Negative Control, Western Blot, Control, Activation Assay, Confocal Microscopy, Two Tailed Test, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: A conformational change within the WAVE2 complex regulates its degradation following cellular activation

doi: 10.1038/srep44863

Figure Lengend Snippet: ( a (i)) Jurkat T cells deficient in VAV-1 (JVAV, upper panel, n = 10 cells), and JVAV cells reconstituted with VAV-1 wt (JVAV/VAV wt, lower panel, n = 16 cells) transfected with a plasmid encoding YFP-WAVE2-CFP were plated over stimulatory coverslips, fixed following 3 min activation, and the FRET efficiency between CFP and YFP was measured. A graph showing the mean FRET efficiency in the indicated cell types expressing YFP-WAVE2-CFP plated over stimulatory coverslips is presented on the right. Graph shows means ± SEM from three independent experiments. Significance was determined by two-tailed Student’s t-test. ( a (ii)) JVAV and JVAV/VAV wt were left unstimulated (−), or were co-stimulated (+) with anti-CD3 and anti-CD28 antibodies. Cell lysates were subjected to immunoprecipitation (IP) with anti-Ub antibody, and samples were analyzed by western blotting (IB) with anti-WAVE2 antibody. Ubiquitylated WAVE2 appears at a molecular weight of 83 kDa. Bottom: WCLs before immunoprecipitation were analyzed by western blotting with the indicated antibodies. All western blots are representative of three independent experiments. ( b ) Scheme of point-mutated YFP-WAVE2-CFP with tyrosine 124 and 150 residues replaced by phenylalanine residues. Jurkat T cells transfected with plasmids encoding YFP-WAVE2-CFP wt (upper panel, n = 43 cells) or YFP-WAVE2-CFP Y124F, Y150F (lower panel, n = 21 cells) were plated over stimulatory coverslips, fixed following 3 min activation, and the FRET efficiency between CFP and YFP was measured. A graph showing the mean FRET efficiency in cells expressing either YFP-WAVE2-CFP wt or YFP-WAVE2-CFP Y124F, Y150F that were plated over stimulatory coverslips is presented on the right. Graph shows means ± SEM from three independent experiments. Significance was determined by two-tailed Student’s t-test.

Article Snippet: Primary antibodies for immunoprecipitation and immunoblotting: mouse anti-GFP, rat anti-HA peroxidase 3F10 (Roche Applied Science); mouse anti-GAPDH (Biodesign); rabbit anti-ubiquitin (Dako); rabbit anti-Hem-1 (Novus); goat anti-WAVE2 D16, mouse anti-ubiquitin P4D1, mouse anti-Vav D7 (Santa Cruz Biotechnology, Inc); rabbit anti-phospho WAVE2 (Ser-351), mouse anti-phosphotyrosine (pTy) 4G10 (Millipore).

Techniques: Transfection, Plasmid Preparation, Activation Assay, Expressing, Two Tailed Test, Immunoprecipitation, Western Blot, Molecular Weight