Journal:

Article Title: Naturally Occurring Truncated trkB Receptors Have Dominant Inhibitory Effects on Brain-Derived Neurotrophic Factor Signaling

doi:

Figure Lengend Snippet: Epitope tags distinguish the ATP-binding mutant from the wild-type trkB receptor. A, COS cells transfected with either vector control (lane 1) or trkBIVH (lane 2). Lysates were immunoprecipitated with IVH Ab (12CA5), separated by 6% SDS-PAGE, then immunoblotted with IVH Ab. B, COS cells transfected with either vector control (lane 1) or ATPmutmyc (lane 2). Lysates were immunoprecipitated with anti-myc Ab (9E10), separated by 6% SDS-PAGE, and then blotted with anti-myc Abs.

Article Snippet: Mouse monoclonal epitope antibodies (Abs) were obtained from Boehringer Mannheim (12CA5-IVH, Indianapolis, IN) or Oncogene Science (9E10-myc).

Techniques: Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page

Journal:

Article Title: Naturally Occurring Truncated trkB Receptors Have Dominant Inhibitory Effects on Brain-Derived Neurotrophic Factor Signaling

doi:

Figure Lengend Snippet: Intermolecular phosphorylation of ATP-binding mutant by wild-type gp145trkB. A, Schematic diagram of epitope-tagged wild-type and mutant trkB receptors. PCR was used to attach an IVH tag to the C terminus of the wild-type trkB receptor (trkBIVH) and myc tag to the C terminus of the ATP-binding mutant (ATPMutmyc) as described in Materials and Methods. Amino acid numbers are listed for the extracellular (EC) or transmembrane domains (TM), ATP-binding site (Lys or Met), and epitope tags (IVH or Myc). B, BDNF induces rapid tyrosine phosphorylation of trkBIVH receptors. COS cells were transfected with vector control (lanes 1, 2) or trkBIVH (lanes 3, 4). Cells in lanes 2 and 4 were stimulated for 5 min with 100 ng/ml BDNF. Lysates were immunoprecipitated with IVH Ab, separated by 6% SDS-PAGE, and then immunoblotted with the APT Ab 4G10. C, Intermolecular tyrosine phosphorylation of ATPMutmyc by trkBIVH. COS cells were transfected with vector alone (lanes 1, 6), ATPMutmyc (lane 2), ATPmutmyc + trkBIVH (lanes 3, 4), or trkBIVH alone (lane 5). Cells in lane 4 were pretreated with 50 mm sodium orthovanadate for 3 hr before lysis. Cells were stimulated with BDNF, then immunoprecipitated with either anti-myc (lanes 1–4) or anti-IVH Abs (lanes 5, 6). Proteins were separated by 6% SDS-PAGE and were then immunoblotted with APT Ab. The location of gp145trkB is indicated by an arrow.

Article Snippet: Mouse monoclonal epitope antibodies (Abs) were obtained from Boehringer Mannheim (12CA5-IVH, Indianapolis, IN) or Oncogene Science (9E10-myc).

Techniques: Phospho-proteomics, Binding Assay, Mutagenesis, Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page, Lysis

Journal: Frontiers in Immunology

Article Title: Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction

doi: 10.3389/fimmu.2020.577838

Figure Lengend Snippet: SVV infection induces transient SG formation relying on viral replication. (A,B) 293T cells were mock-infected or infected with SVV (MOI = 1) at the indicated time points. And cells were treated with SA (0.5 mM) for 50 min as a positive control. The cells were fixed and stained with either rabbit polyclonal specific antibodies for G3BP1 (green) and mouse monoclonal specific antibodies for VP3 (red) or either rabbit polyclonal specific antibodies for eIF4GI (green) and mouse monoclonal specific antibodies for VP3 (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. Then cells were analyzed by confocal microscopy. (C) The percentage of cells containing SGs was calculated in three independent experiments. At least 100 cells were counted each time. Data are represented as means ± SD. Student's t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. (D) 293T cells were infected with SVV or UV-inactivated SVV (MOI = 1) for 4 h. The cells were analyzed by confocal microscopy after staining with anti-VP3 antibodies and anti-G3BP1 antibodies.

Article Snippet: Mouse monoclonal or rabbit polyclonal antibodies against Flag tag (M385-3L) and HA tag (M180-3) were purchased from Medical and Biological Laboratories (MBL).

Techniques: Infection, Positive Control, Staining, Confocal Microscopy

Journal: Frontiers in Immunology

Article Title: Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction

doi: 10.3389/fimmu.2020.577838

Figure Lengend Snippet: SVV triggers SG formation via PKR-eIF2α signaling pathway. (A) 293T cells were mock-infected or infected with SVV at a MOI of 1 at the indicated time point. The cell lysates were analyzed by western blot using anti-VP1, anti-eIF2α, anti-phosphorylated eIF2α, and anti-α-tubulin antibodies. (B) 293T cells were transducted with shPKR lentivirus and shNC lentivirus for 48 h and then infected with SVV at 1 MOI for 4 h. The cells were fixed and stained with either rabbit polyclonal specific antibodies for G3BP1 (green) and mouse monoclonal specific antibodies for VP3 (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (C) The percentage of cells containing SGs was calculated in three independent experiments. At least 100 cells were counted each time. (D) 293T cells were transducted with shPKR lentivirus and shNC lentivirus for 48 h and then infected with SVV at 1 MOI for 8 h. The cell lysates were analyzed by western blot using anti-VP1, anti-PKR, anti-eIF2α, anti-phosphorylated eIF2α, and anti-α-tubulin antibodies. (E) Totol RNA was extracted and the relative amount of mRNA of PKR was determined by RT-qPCR and GADPH was considered as housekeeping genes. (F) The supernatants were harvested and the virus titers were determined by plaque assay. Data are represented as means ±SD. Student's t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant.

Article Snippet: Mouse monoclonal or rabbit polyclonal antibodies against Flag tag (M385-3L) and HA tag (M180-3) were purchased from Medical and Biological Laboratories (MBL).

Techniques: Infection, Western Blot, Staining, Quantitative RT-PCR, Virus, Plaque Assay

Journal: Frontiers in Immunology

Article Title: Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction

doi: 10.3389/fimmu.2020.577838

Figure Lengend Snippet: Inhibition of SG formation has no significant effect on SVV replication. (A) 293T cells were transducted with shTIA1 lentivirus and shNC lentivirus for 48 h and then infected with SVV at 1 MOI for 4 h. The cells were fixed and stained with either rabbit polyclonal specific antibodies for G3BP1 (green) and mouse monoclonal specific antibodies for VP3 (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (B) The percentage of cells containing SGs was calculated in three independent experiments. At least 100 cells were counted each time. (C) 293T cells were transducted with shG3BP1 lentivirus and shNC lentivirus for 48 h and then infected with SVV at 1 MOI for 4 h. The cells were fixed and stained with either rabbit polyclonal specific antibodies for eIF4GI (green) and mouse monoclonal specific antibodies for VP3 (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (D) The percentage of cells containing SGs was calculated in three independent experiments. (E,F) 293T cells were transducted with shTIA1 lentivirus and shNC lentivirus for 48 h and then infected with SVV at 1 MOI for 6 h. The cell lysates were analyzed by western blot using anti-TIA1, anti-VP1, and anti-α-tubulin antibodies. The supernatants were harvested and the virus titers were determined by plaque assay. (G,H) 293T cells were transducted with shG3BP1 lentivirus and shNC lentivirus for 48 h and then infected with SVV at 1 MOI for 6 h. The cell lysates were analyzed by western blot using anti-G3BP1, anti-VP1, and anti-α-tubulin antibodies. The supernatants were harvested and the virus titers were determined by plaque assay. (I) 293T cells were transfected with vector, HA-eIF2α, or HA-eIF2α-S51A for 20 h and then infected with SVV at 1 MOI for another 6 h. The cell lysates were analyzed by western blot using anti-VP1, anti-TIA1, and anti-α-tubulin antibodies. (J) The supernatants were harvested and the virus titers were determined by plaque assay. Data are represented as means ±SD. Student's t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant.

Article Snippet: Mouse monoclonal or rabbit polyclonal antibodies against Flag tag (M385-3L) and HA tag (M180-3) were purchased from Medical and Biological Laboratories (MBL).

Techniques: Inhibition, Infection, Staining, Western Blot, Virus, Plaque Assay, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction

doi: 10.3389/fimmu.2020.577838

Figure Lengend Snippet: SVV 3C inhibits SA-induced SG formation depending on its protease activity. (A) 293T cells were infected or uninfected with SVV at 1 MOI for 9 h and subsequently treated with 0.5 mM SA for another 50 min. The cells were fixed and stained with either rabbit polyclonal specific antibodies for G3BP1 (green) and mouse monoclonal specific antibodies for VP3 (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (B) The percentage of cells containing SGs was calculated in three independent experiments. At least 100 cells were counted each time. (C) 293T cells were transiently transfected with vector or HA-3C for 20 h and then stimulated with heat shock at 45°C for 50 min or poly I:C for 12 h. The cells were fixed and stained with either rabbit polyclonal specific antibodies for G3BP1 (red) and mouse monoclonal specific antibodies for HA (green). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. (D) The percentage of 3C-positive cells containing SGs was calculated in three independent experiments. At least 100 cells were counted each time. (E) 293T cells were transiently transfected with vector, HA-3C, or HA-3C variants for 20 h and then treated with 0.5 mM SA for another 50 min. (F) The percentage of 3C- or 3C mutants-positive cells containing SGs was calculated in three independent experiments. At least 100 cells were counted each time. Data are represented as means ± SD. Student's t -test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant.

Article Snippet: Mouse monoclonal or rabbit polyclonal antibodies against Flag tag (M385-3L) and HA tag (M180-3) were purchased from Medical and Biological Laboratories (MBL).

Techniques: Activity Assay, Infection, Staining, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Seneca Valley Virus 3C Protease Inhibits Stress Granule Formation by Disrupting eIF4GI-G3BP1 Interaction

doi: 10.3389/fimmu.2020.577838

Figure Lengend Snippet: SVV 3C inhibits SG formation by disrupting G3BP1-eIF4GI interaction. (A) 293T cells were transfected with vector or HA-3C for 12 h and subsequently transfected with poly I:C for another 12 h. The cell lysates were analyzed by western blot using anti-VP1, anti-phosphorylated PKR, anti-PKR, anti-eIF2α, anti-phosphorylated eIF2α, and anti-α-tubulin antibodies. (B) 293T cells were cotransfected with SVV 3C or EV71 3C and G3BP1 for 24 h. The cell lysates were analyzed by western blot using anti-Flag, anti-HA, and anti-α-tubulin antibodies. (C) 293T cells were cotransfected with G3BP1, G3BP2, TIA1, or TIAR and SVV 3C for 24 h. Subsequently, the interaction between G3BP1, G3BP2, TIA1, or TIAR and SVV 3C was measured by anti-Flag immunoprecipitation. (D) 293T cells were infected or uninfected with SVV at 1 MOI for 8 h, and the cell lysates were subjected to immunoprecipitation with anti-eIF4GI antibodies. (E) 293T cells were transfected with vector, 3C, or 3C-DM and eIF4GI for 24 h and then infected or uninfected with SVV at 1 MOI for 8 h. And the cell lysates were subjected to immunoprecipitation with anti-eIF4GI antibodies.

Article Snippet: Mouse monoclonal or rabbit polyclonal antibodies against Flag tag (M385-3L) and HA tag (M180-3) were purchased from Medical and Biological Laboratories (MBL).

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Infection