Journal: Nature Communications

Article Title: Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration

doi: 10.1038/s41467-017-01107-0

Figure Lengend Snippet: NEDD9 as DLC1 interacting protein is required for the polarized expression of DLC1. a Venn diagram showing the number of proteins enriched by DLC1. b DLC1-associated proteins involved in major canonical pathways as assessed by the Ingenuity Pathway Knowledge base. c Interactome network of DLC1 and its associated proteins involved in cell motility and cytoskeleton regulation. d Immunoprecipitation (IP) was performed in neural tube lysate using anti-DLC1, and IgG as control. Immunoprecipitated proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and Western blotting was performed with anti-NEDD9 and anti-DLC1 antibodies. e Schematic showing the design of the C-terminally truncated DN-DLC1 constructs. IP with anti-V5 was performed in lysates extracted from neural tubes electroporated with the indicated constructs followed by western blot with anti-V5 or anti-NEDD9. White arrowhead indicates the specific protein band of DN-DLC1 (SAM). f In situ hybridization for NEDD9 , DLC1 , SOX9 and SOX10 on transverse sections of st 11 chick embryo. Immunofluorescence for NEDD9, SOX9 and SOX10 on transverse sections of st 11 chick embryo. Scale bar: 50 µm. g Immunofluorescence for NEDD9 and phalloidin on emigrating NCCs from neural tube explants and nuclei are stained with DAPI. Polarized expression of NEDD9 is shown in pseudocolour. Line scan analysis showing the average fluorescence intensity of NEDD9 along the white dotted line ( n = 103/11 explants). h Immunofluorescence for DLC1 and phalloidin on NCCs expressing control morpholino (Ctrl-MO) ( n = 65/11 explants) or NEDD9-MO ( n = 112/12 explants). Distribution of DLC1 expression in both treatments are shown in pseudocolour. Scale bars, 20 µm. i Line scan analysis showing the average fluorescence intensity of DLC1 in NEDD9-MO-treated NCCs ( n = 112/12 explants)

Article Snippet: Horseradish peroxidase-conjugated anti-mouse immunoglobulin G (GE Healthcare) was used for the second reaction at 1:5000 dilutions.

Techniques: Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Construct, In Situ Hybridization, Immunofluorescence, Staining, Fluorescence

Journal: Nature Communications

Article Title: Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration

doi: 10.1038/s41467-017-01107-0

Figure Lengend Snippet: SOXE factors regulate DLC1 and NEDD9 expression. a In situ hybridization for DLC1 on transverse sections of embryos electroporated with SOX10 at 12 hpt ( n = 9). Scale bar, 50μm. b In situ hybridization for DLC1 on embryos electroporated with Ctrl-MO ( n = 10) or SOX10-MO ( n = 10) at 24hpt. Black arrows indicate loss of DLC1 expression on the transfected side of the neural tube. Scale bar, 10μm. c In situ hybridization for FOXD3 on embryos electroporated with SOX9 ( n = 9) or SOX9 + DN-DLC1 ( n = 9) at 24hpt. Scale bar, 20 µm d In situ hybridization for FOXD3 on embryos electroporated with SOX10 ( n = 10) or SOX10 + DN-DLC1 ( n = 10) at 24hpt. Scale bar, 10 µm. e Immunofluorescence for NEDD9 and in situ hybridization for NEDD9 on transverse sections of embryos electroporated with SOX9 ( n = 12) at 6 hpt. Scale bar, 50 µm. f Immunofluorescence for SOX9 and in situ hybridization for NEDD9 on transverse sections of embryos electroporated with Ctrl-MO ( n = 9) or SOX9-MO ( n = 9). White arrowhead indicates loss of SOX9 expression on the transfected side. Scale bar: 50 µm. g Schematic diagram showing the seven exons ( black ) of chick NEDD9 gene with promoter region in blue, −9.5 kb enhancer 1 (E1 harboring two putative SOX9 binding motifs, B1 and B2, and + 45 kb enhancer 2 (E2) containing a putative SOX9 binding motif, B3. h Fold enrichments of three independent ChIP-qPCR assays for SOX9, SOX10 and IgG (control) antibodies on B1, B2 and B3. Data represented as fold enrichments for putative SOX9 binding motifs relative to control. Mean±s.e.m. Bonferroni multiple comparison test, * p

Article Snippet: Horseradish peroxidase-conjugated anti-mouse immunoglobulin G (GE Healthcare) was used for the second reaction at 1:5000 dilutions.

Techniques: Expressing, In Situ Hybridization, Transfection, Immunofluorescence, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Globular-shaped variable lymphocyte receptors B antibody multimerized by a hydrophobic clustering in hagfish

doi: 10.1038/s41598-018-29197-w

Figure Lengend Snippet: Characterization of multimerized VLRB by hydrophobic clustering in hagfish plasma. ( A ) Total hagfish plasma proteins (3.5 μg/lane) were separated by 8% native-PAGE (left) or 12% SDS-PAGE (right) under non-reducing conditions, followed by Western blot analysis with 11G5. ( B ) Western blot analysis of VLRB in total hagfish plasma proteins treated with SDS (0, 0.003, 0.01, 0.03, 0.1 or 0.3%, v/v) at room temperature for 10 min. ( C ) Hagfish plasma samples were incubated with NP-40 (0.01, 0.03, or 0.1%, v/v), Triton X-100 (0.005, 0.015, or 0.03%, v/v), or SDS (0.003, 0.01, or 0.03%, v/v) at room temperature for 10 min. The samples were resolved by native-PAGE (8%), and VLRBs were detected with 11G5 followed by goat anti-mouse IgG-HRP.

Article Snippet: The blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Thermo Scientific), and the results were visualized using a SuperSignal West Pico Chemiluminescent Substrate kit (#34080, Thermo Scientific).

Techniques: Clear Native PAGE, SDS Page, Western Blot, Incubation

Journal: PLoS Pathogens

Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

doi: 10.1371/journal.ppat.1006766

Figure Lengend Snippet: Phospholipid scramblase is required for phosphatidylserine and Akt relocalization. (A) CaSki cells were mock-infected or infected with HSV-2(G) and 15 minutes, 30 minutes, 1 hour and 4 hours post-infection, cell lysates were harvested. Lysates were incubated with a goat anti-PLSCR1 antibody and immune complexes precipitated with protein A-agarose and analyzed by Western blotting with a mouse anti-phosphotyrosine (PY20) or mouse anti-PLSCR1 mAb. The blot is representative of results obtained in 2 independent experiments. (B). CaSki, VK2E6/E7 or HaCAT cells were transfected with siRNA targeting PLSCR1 or a control siRNA (siCtrl) and protein expression was evaluated by Western blot probing for PLSCR1 (rabbit anti-PLSCR1) and β-actin (mouse monoclonal). Blot is representative of results obtained in 3 independent experiments. (C) CaSki or HaCAT cells were transfected with siControl (siCtrl) or siPLSCR1 RNA and 72 h post-transfection, plasma membranes were stained with Alexa Fluor 594-conjugated wheat germ agglutinin (red) and then synchronously infected with HSV-1(KOS), HSV-2(G), or mock-infected (4 hours at 4°C, washed, and then shifted to 37°C for 15 min and treated with low pH citrate buffer). The cells were then fixed and nuclei were stained blue with Hoechst, and phosphatidylserines (PtdS) or PLSCR1 stained green with respective primary murine and secondary Alexa 488-conjugated secondary antibodies. Images are representative of results obtained from 2–3 independent experiments. (D). CaSki cells were transfected with the indicated siRNA and then synchronously infected with HSV-2(G), fixed with or without Triton X permeabilization and stained with fluorescently-conjugated antibodies to Akt (red) and PLSCR1 (green); nuclei were stained blue with DAPI. Representative 3-D images from 3 independent experiments are shown; bars = 10μm.

Article Snippet: The secondary antibodies for Western blots were horseradish peroxidase-conjugated goat anti-mouse (170–5047, Bio-Rad, Hercules, CA), goat anti-rabbit (170–5046, Bio-Rad), and donkey anti-goat 1:1000 (sc-2020, Santa Cruz).

Techniques: Infection, Incubation, Western Blot, Transfection, Expressing, Staining