Journal: The EMBO Journal

Article Title: Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2

doi: 10.15252/embj.2020105139

Figure Lengend Snippet: A, B MARCH2 +/+ HEK293T cells transfected with Flag‐tagged empty vector or MARCH2 −/− HEK293T cells transfected with Flag‐tagged empty vector or MARCH2 WT were infected with VSV‐GFP (MOI = 0.5). (A) Viral replication was determined at 24 hpi by fluorescence microscopy, fluorescence absorbance, and plaque assay. (B) Concentration of IFN‐β and IL‐6 secreted in supernatants was determined at 12 and 24 hpi by ELISA, Scale bar, 50 μm. C IFN‐β luciferase reporter assay. MARCH2 +/+ or MARCH2 −/− HEK293T cells were transfected with firefly luciferase reporter plasmid encoding INF‐β promoter, TK‐Renilla plasmid, and expression plasmids of RIG‐I 2CARD, MAVS, or TBK‐1. MARCH2 −/− HEK293T cells were reconstituted with increasing amount of Flag‐tagged MARCH2 plasmid (100, 200 ng). D MARCH2 +/+ , MARCH2 −/− , or MARCH2 −/− HEK293T reconstituted with Flag‐tagged MARCH2 WT were infected with VSV‐GFP (MOI = 0.5). Cells were harvested at indicated hours post‐infection; total and phosphorylated TBK1, IRF3, and p65 were measured by immunoblotting in whole‐cell lysates. β‐actin was used to confirm equal loading of protein. Data information: * P < 0.05, ** P < 0.01 (two‐tailed Student's t‐ test). Data are representative of at least two independent experiments, each with similar results, and expressed as the mean ± SD of two biological replicates. Source data are available online for this figure.

Article Snippet: The replication of GFP‐tagged virus was measured using fluorescence modulator (GloMax‐Multi Detection System, Promega) and determined by standard plaque assay.

Techniques: Transfection, Plasmid Preparation, Infection, Fluorescence, Microscopy, Plaque Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Expressing, Western Blot, Two Tailed Test

Journal: The EMBO Journal

Article Title: Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2

doi: 10.15252/embj.2020105139

Figure Lengend Snippet: A, B NEMO degradation assay. (A) HEK293T cells were transfected with different doses of Strep‐tagged MARCH2 in the presence of HA‐tagged NEMO plasmid. (B) MARCH2 +/+ and MARCH2 −/− HEK293T cells were infected with PR8‐GFP (MOI = 1) in time‐dependent manner. Cells were harvested following infection; NEMO expression level, total and phosphorylated IRF‐3, and TBK‐1 were measured by immunoblotting. β‐actin was used to confirm equal loading of protein. C HEK293T cells transfected with a Strep‐tagged empty vector or MARCH2 together with GST‐tagged NEMO were treated with MG132 (10 μM), chloroquine, NH 4 Cl, or Z‐VAD for 6 h before harvest. Whole‐cell lysates were subjected to immunoblotting with indicated antibodies. D HEK293T cells were transfected with different doses of Strep‐tagged MARCH2 together with GST‐tagged NEMO plasmid. MG132 (10 μM) was added to cells for 6 h prior to harvest. Whole‐cell lysates were subjected to immunoblotting with the indicated antibodies. E Confocal microscopy was used to examine co‐localization of MARCH2 and NEMO in HeLa cells upon NDV infection (MOI = 1) in the presence or absence of MG132 (10 μM). Scale bar, 10 μm. Arrow indicates the co‐localized NEMO and MARCH2 protein. F HEK293T cells transfected with Strep‐tagged empty vector or MARCH2 with GST‐tagged NEMO and each different HA‐tagged ubiquitin mutants (indicated lysine (K) only, other lysines (K) mutated to arginines (R)) were treated with MG132 (10 μM) for 6 h before harvest. Lysates were subjected to pull‐down with GST beads, followed by immunoblotting with an anti‐HA antibody. Whole‐cell lysates were determined by immunoblotting with the indicated antibodies. G NEMO ubiquitination assay. HEK293T cells were transfected with different doses of Strep‐tagged MARCH2 in the presence of a HA‐tagged NEMO plasmid. Lysates were immunoprecipitated with anti‐HA antibody followed by immunoblotting with anti‐K48 antibody. H–J MARCH2 +/+ HEK293T cells harboring empty vector or MARCH2 −/− HEK293T cells harboring empty vector, Flag‐tagged MARCH2, or MARCH2 CCH (a mutant harboring mutations in residues within the catalytic domain: C64S, C67S, and H90Q) were infected with VSV‐GFP (MOI = 0.05). Viral replication was assessed under a fluorescence microscope (H) or by measuring GFP expression using a fluorescence modulator (I, top) and in a plaque assay (I, bottom). (J) Levels of IFN‐β (top) and IL‐6 (bottom) in cell supernatants were measured in an ELISA. K In vitro ubiquitination assay for NEMO. HEK293T cells were transfected with GST‐tagged NEMO, Strep‐tagged MARCH2, or MARCH2 CCH individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti‐K48 linkage‐specific polyubiquitin antibody. Data information: * P < 0.05, ** P < 0.01 (two‐tailed Student's t‐ test). Data are representative of at least two independent experiments, each with similar results, and are expressed as the mean ± SD of three biological replicates. Source data are available online for this figure.

Article Snippet: The replication of GFP‐tagged virus was measured using fluorescence modulator (GloMax‐Multi Detection System, Promega) and determined by standard plaque assay.

Techniques: Degradation Assay, Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Confocal Microscopy, Ubiquitin Proteomics, Immunoprecipitation, Mutagenesis, Fluorescence, Microscopy, Plaque Assay, Enzyme-linked Immunosorbent Assay, In Vitro, Incubation, Two Tailed Test

Journal: The EMBO Journal

Article Title: Negative regulation of NEMO signaling by the ubiquitin E3 ligase MARCH2

doi: 10.15252/embj.2020105139

Figure Lengend Snippet: A HEK293T cells were transfected with GST‐tagged NEMO‐WT and its point mutants with or without Strep‐tagged MARCH2. Whole‐cell lysates were subjected to immunoblotting with the indicated antibodies. B HEK293T cells were transfected with GST‐tagged NEMO‐WT and its point mutants together with Strep‐tagged MARCH2 in the presence of MG132. Whole‐cell lysates were subjected to a GST‐PD assay, followed by immunoblotting with an anti‐K48 linkage‐specific polyubiquitin antibody. C In vitro ubiquitination assay for NEMO‐K326R. HEK293T cells were transfected with GST‐tagged NEMO, GST‐tagged NEMO‐K326R, or Strep‐tagged MARCH2 individually. Immunoprecipitates were incubated with a reaction mixture containing ubiquitin, E1, and UbcH6 (E2), followed by immunoblotting with an anti‐K48 linkage‐specific polyubiquitin antibody. D–G HEK293T cells transfected with GST‐tagged empty vector, NEMO‐WT, or NEMO‐K326R, with or without Strep‐tagged MARCH2 were infected with VSV‐GFP. (D‐F) Viral replication was determined by fluorescence microscopy (D), fluorescence absorbance (E), and a plaque assay (F). (G) IFN‐β and IL‐6 in the cell supernatants were measured in an ELISA. H, I IFN‐β (H) and NF‐κB (I) luciferase reporter assay. Data information: ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001 (two‐tailed Student's t‐ test). Data are representative of at least two independent experiments, each with similar results, and expressed as the mean ± SD of three biological replicates. Source data are available online for this figure.

Article Snippet: The replication of GFP‐tagged virus was measured using fluorescence modulator (GloMax‐Multi Detection System, Promega) and determined by standard plaque assay.

Techniques: Transfection, Western Blot, In Vitro, Ubiquitin Proteomics, Incubation, Plasmid Preparation, Infection, Fluorescence, Microscopy, Plaque Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Two Tailed Test

Journal: Frontiers in Plant Science

Article Title: Chloroplast Electron Chain, ROS Production, and Redox Homeostasis Are Modulated by COS-OGA Elicitation in Tomato ( Solanum lycopersicum ) Leaves

doi: 10.3389/fpls.2020.597589

Figure Lengend Snippet: Normalized apoplastic reactive oxygen species (ROS) production in tomato leaf disks. Tomato leaf disks were treated with 0.1% Tween 20 (control) or chitooligosaccharides–oligogalacturonides (COS-OGA; 62.5 mg/l). The average apoplastic ROS production of the COS-OGA-treated leaf disks is expressed as a percentage luminol chemiluminescence emitted in comparison to the control ± standard deviation ( n = 12). ***indicates a statistically significant difference to control according to Student’s t test ( p < 0.001, R ).

Article Snippet: Apoplastic ROS were measured using GloMax-Multi + Detection system (Promega, United States) using a protocol modified from an earlier described protocol ( ).

Techniques: Control, Comparison, Standard Deviation