monoclonal rabbit anti wave2 antibody  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Differential Role of the RAC1-Binding Proteins FAM49b (CYRI-B) and CYFIP1 in Platelets.

doi: 10.3390/cells13040299

Figure Lengend Snippet: Figure 6. Localization of WAVE2 in Fam49b−/−platelets. (A) Expression of WAVE2 and ARPC2 in control and mutant platelets was assessed by Western blot analysis. α-Tubulin served as loading control (n = 3). (B) Representative confocal fluorescence images of Cyfip1−/−and Fam49b−/−- platelets stained for WAVE2. The dotted line in the Cyfip1−/−image indicates the cell perimeter. (C) Super-resolved two-color dSTORM images of WAVE2 (green) and F-actin (magenta) in control- and Fam49b−/−-platelets and magnifications of the boxed areas. Scale bars: 3 µm in (B); 2 µm in (C), 200 nm in magnifications of (C).

Article Snippet: Subsequently, the samples were immunolabeled for 1 h with monoclonal rabbit anti-WAVE2 antibody (#3659, Cell Signaling) diluted at 1:200 in PBS containing 5% BSA.

Techniques: Expressing, Control, Mutagenesis, Western Blot, Fluorescence, Staining

Journal: bioRxiv

Article Title: Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

doi: 10.1101/443309

Figure Lengend Snippet: (A-B) OT1 CTLs expressing WASP-GFP or WAVE2-GFP as indicated were imaged by confocal microscopy on fluorescent micropillars bearing H2-K b -OVA and ICAM1. (A) Time-lapse montages of representative CTLs, with micropillars shown in red. z-projection images (top views) are shown above with corresponding sagittal views below. Cyan dotted lines denote the slicing planes used for the sagittal images. Magenta arrowheads indicate representative lateral accumulations of WAVE2-GFP. (B) Centralization factor analysis of WASP-GFP and WAVE2-GFP (see Materials and Methods), with time 0 denoting initial contact with the pillars. N = 6 for each cell type. (C-D) NT-CR, WASP-CR, and WAVE2-CR CTLs expressing Lifeact-GFP were imaged by confocal microscopy on fluorescent micropillars bearing H2-K b -OVA and ICAM1. (C) Time-lapse montages of representative CTLs, with micropillars shown in red. z-projection images (top views) are shown above with corresponding sagittal views below. Cyan dotted lines denote the slicing planes used for the sagittal images. (D) Centralization factor analysis of Lifeact-GFP in NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs, with time 0 denoting initial contact with the pillars. N = 6 for each cell type. In all montages, time in MM:SS is indicated in the upper left corner of each top view image. Scale bars = 2 μm. In graphs, filled white diamonds and filled red diamonds indicate P < 0.05 and P < 0.01, respectively, calculated by two-tailed Student’s T-test comparing WASP-GFP to WAVE2-GFP (B) or WASP-CR and WAVE2-CR to NT-CR (C). Error bars denote SEM.

Article Snippet: Suppression of PTEN, WASP and WAVE2 was confirmed using the following antibodies: anti-PTEN monoclonal antibody (clone D4.3; Cell Signaling Technology), anti-WASP monoclonal antibody (clone B-9; Santa Cruz) and anti-WAVE2 monoclonal antibody (clone D2C8; Cell Signaling Technology).

Techniques: Expressing, Confocal Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

doi: 10.1101/443309

Figure Lengend Snippet: (A) Immunoblot analysis of WASP and WAVE2 expression in NT-CR, WASP-CR, and WAVE2-CR CTLs. Actin served as a loading control. (B) Diagram schematizing the radial distance histogram analysis of pillar deflections shown in . (C) RMA-s target cells were loaded with increasing concentrations of OVA and then mixed with NT-CR, WASP-CR, or WAVE2-CR CTLs. Specific lysis of RMA-s cells is shown, with error bars denoting SEM. This experiment highlights the cytotoxicity defect occasionally observed in WAVE2-CR CTLs at high OVA concentrations. (D) Adhesion of fluorescently labeled NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs to wells coated with the indicated concentrations of ICAM1 in the absence (left) or presence (right) of H2-K b -OVA (pMHC). Error bars denote SEM. (E) NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs were loaded with Fura2-AM Ca 2+ dye and then imaged on stimulatory glass surfaces coated with H2-K b -OVA and ICAM1. Ca 2+ signaling was analyzed by quantifying mean normalized Fura2 ratio over time (see Materials and Methods). N ≥ 29 for each cell type, with error bars denote SEM. In C, D, and E, filled white diamonds and filled red diamonds indicate P < 0.05 and P < 0.01, respectively, calculated by two-tailed Student’s T-test comparing WASP-CR and WAVE2-CR to NT-CR. (F) NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs were stimulated with beads coated with H2-K b -OVA and ICAM1 for the indicated times and then lysed. pErk1/2, pAKT, and IκB levels were assessed by immunoblot using actin as a loading control.

Article Snippet: Suppression of PTEN, WASP and WAVE2 was confirmed using the following antibodies: anti-PTEN monoclonal antibody (clone D4.3; Cell Signaling Technology), anti-WASP monoclonal antibody (clone B-9; Santa Cruz) and anti-WAVE2 monoclonal antibody (clone D2C8; Cell Signaling Technology).

Techniques: Western Blot, Expressing, Control, Lysis, Labeling, Two Tailed Test

Journal: bioRxiv

Article Title: Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

doi: 10.1101/443309

Figure Lengend Snippet: (A-B) NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs were labeled with a fluorescent anti-CD45 F ab and imaged on narrow fluorescent micropillars coated with H2-K b -OVA and ICAM1. (A) Time-lapse montages of representative CTLs showing pillar deflection. Time in M:SS is indicated in the upper left corner of each top view image. Scale bars = 2 μm. (B) Total force exertion against pillar arrays was graphed versus time. Color bar above each graph indicates the P-value for each time point (two-tailed Student’s T-test). (C) Histogram showing the distribution of strong deflections as a function of radial distance from the center of the IS. N = 10 for each cell type in B and C. (D-F) RMA-s target cells were loaded with increasing concentrations of OVA and then mixed with NT-CR, WASP-CR, or WAVE2-CR CTLs. (D) Specific lysis of RMA-s cells. (E) Degranulation measured by surface exposure of Lamp1. (F) CTL-target cell conjugate formation measured by flow cytometry. All error bars denote SEM. In D-F, filled white diamonds and filled red diamonds indicate P < 0.05 and P < 0.01, respectively, calculated by two-tailed Student’s T-test comparing WASP-CR and WAVE2-CR to NT-CR.

Article Snippet: Suppression of PTEN, WASP and WAVE2 was confirmed using the following antibodies: anti-PTEN monoclonal antibody (clone D4.3; Cell Signaling Technology), anti-WASP monoclonal antibody (clone B-9; Santa Cruz) and anti-WAVE2 monoclonal antibody (clone D2C8; Cell Signaling Technology).

Techniques: Labeling, Two Tailed Test, Lysis, Flow Cytometry

Journal: bioRxiv

Article Title: Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

doi: 10.1101/443309

Figure Lengend Snippet: (A) Schematic diagram of a CTL inducing distortion of an adherent target cell. (B-C) NT-CR, WASP-CR, and WAVE2-CR OT1 CTLs expressing Lifeact-GFP were applied to confluent cultures of OVA-loaded endothelial target cells expressing iRFP670 and then imaged using lattice light-sheet microscopy. (B) Left, time-lapse montages of representative “vertically” oriented synapses, with z-projection images (top views) shown above and corresponding sagittal views below. Cyan dotted lines denote the slicing planes used for the sagittal images. In z-projection images, target cells are visualized by surface representation. Two z-projections are shown for each time point; Lifeact-GFP is shown on the left and the outline of the CTL of interest on the right. Time in M:SS is indicated in the upper left corner of each sagittal image. Scale bars = 2 μm. Yellow arrowheads denote protrusive structures in the NT-CR CTL that invade the target cell space. Right, target IS volume (see Materials and Methods) graphed against time, with time 0 denoting IS initiation. Each line corresponds to one CTL-target cell conjugate. (C) Graph of minimum target IS volume values achieved during the first 400 s of conjugate formation. N = 7 for each cell type. Error bars denote SEM. P calculated by two-tailed Student’s T-test.

Article Snippet: Suppression of PTEN, WASP and WAVE2 was confirmed using the following antibodies: anti-PTEN monoclonal antibody (clone D4.3; Cell Signaling Technology), anti-WASP monoclonal antibody (clone B-9; Santa Cruz) and anti-WAVE2 monoclonal antibody (clone D2C8; Cell Signaling Technology).

Techniques: Expressing, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Interfacial actin protrusions mechanically potentiate killing by cytotoxic T cells

doi: 10.1101/443309

Figure Lengend Snippet: A schematic diagram of the cytolytic IS showing peripheral WAVE2-dependent protrusions and central WASP-dependent protrusions. Red arrows denote force exertion.

Article Snippet: Suppression of PTEN, WASP and WAVE2 was confirmed using the following antibodies: anti-PTEN monoclonal antibody (clone D4.3; Cell Signaling Technology), anti-WASP monoclonal antibody (clone B-9; Santa Cruz) and anti-WAVE2 monoclonal antibody (clone D2C8; Cell Signaling Technology).

Techniques: