Journal: Scientific Reports

Article Title: A fixation method for the optimisation of western blotting

doi: 10.1038/s41598-019-43039-3

Figure Lengend Snippet: Fixation-dependent differences in lectin staining intensities when using PVDF and nitrocellulose membranes. Pooled human serum proteins (3 μg) were separated on 10% SDS-PAGE, and the proteins were transferred onto PVDF and nitrocellulose membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments and followed by staining with lectins (LCA and SNA). Lane i, CBB staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, sample heating at 100 °C; lane v, organic solvent (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) treatments at room temperature; lane vi, organic solvent treatments followed by sample heating at 100 °C. All treatments were applied for 30 min. Left, WB pattern; right, quantitative analysis (n = 3 individual experiments). The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. ** Significantly different p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).

Article Snippet: Biotinylated lectins, LCA, SNA, PHA-E, PHA-L, and AAL (Supplementary Table ), were purchased from Vector Laboratories Inc. (Burlingame, CA, USA).

Techniques: Staining, SDS Page, Software

Journal: PLoS Genetics

Article Title: The steroid hormone 20-hydroxyecdysone binds to dopamine receptor to repress lepidopteran insect feeding and promote pupation

doi: 10.1371/journal.pgen.1008331

Figure Lengend Snippet: A . Apoptosis signal in HaEpi cells after DMSO, 20E (5 μM), dsGFP +20E (5 μM), dsDopEcR+ 20E (5 μM) and DA (10 μM) +20E (5 μM) treatment by using the NucView caspase-3 activity assay kit. a . The ratio of apoptotic cells (green) to the total cells (blue) in the field view was obtained. B . Proliferation signal in HaEpi cells after DMSO, 20E (5 μM), dsGFP +20E (5 μM), dsDopEcR+ 20E (5 μM) and DA (10 μM) +20E (5 μM) treatment by using the 5-ethynyl-2′-deoxyuridine (EdU) kit (Ribobio, Guangzhou, China). b . The ratio of proliferation cells (green) to the total cells (blue) in the field view was obtained. DAPI stained the nucleus (blue). Statistical analysis using the data from 100 × 3 cells. The yellow bars represent 100 μM. C . Western blot analysis of 20E (5 μM) or DA (10 μM) induced proteins phosphorylation for 30 min. a. b . and c . AKT-RFP-His, AKT -RFP-His, and CDK10-RFP-His. 7.5% SDS-PAGE gel. Error bars show the mean ± SD of three times’ biological repetition. ImageJ software was used to transform the image data. Significant differences were calculated by Student’s t test (* p < 0.05; ** p < 0.01) or one-way analysis of variance (ANOVA, p < 0.05).

Article Snippet: The NucView 488 caspase-3 assay kit (NO. 30029 Biotium, Hayward, USA) was used to detect the activity of caspase-3 in the HaEpi cells, according to the manufacturer’s instructions.

Techniques: Caspase-3 Activity Assay, Staining, Western Blot, SDS Page, Software

Journal: Frontiers in Immunology

Article Title: The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-β Production by Targeting TNF Receptor-Associated Factor 3

doi: 10.3389/fimmu.2017.00779

Figure Lengend Snippet: NS1/126-225 inhibits the activation of IRF3. (A) 293T cells were transfected with an IRF3 reporter plasmid and pRL-TK in the presence or absence of RIG-I(N). Cells were cotransfected with plasmids expressing NS1 or NS1/126-225, or an empty vector as a control. Luciferase activity within lysates was determined 24 h posttransfection. (B,C) 293T cells were transfected with an empty vector, NS1, or NS1/126-225 along with or without RIG-I(N) for 24 h. Lysates were then analyzed for levels of IRF3 dimers by western blotting using non-reduced SDS-PAGE (B) , and levels of p-IRF3(S396) and total IRF3 in the cytoplasmic and nuclear lysates (C) . GAPDH and Lamin A/C were used as cytoplasmic and nuclear fraction markers, respectively. (D) A549 cells were transfected with empty vector, NS1, or NS1/126-225 for 24 h. IFN-β production in response to RIG-I(N) was measured by ELISA. (E) Representative fluorescence micrographs of VSV-GFP in 293T cells transfected with an empty vector, NS1, or NS1/126-225 along with RIG-I(N) for 24 h followed by infection with VSV-GFP for 12 h. All experiments were performed at least three times with similar results. ** P < 0.01 by Student’s t -test.

Article Snippet: Then 100 μl of the cleared medium or the IFN-β standard was used in duplicate for detection of IFN-β using a human IFN-β ELISA Kit (Elabscience, E-EL-H0085c), according to the manufacturer’s instructions.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Control, Luciferase, Activity Assay, Western Blot, SDS Page, Enzyme-linked Immunosorbent Assay, Fluorescence, Infection

Journal: Frontiers in Immunology

Article Title: The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-β Production by Targeting TNF Receptor-Associated Factor 3

doi: 10.3389/fimmu.2017.00779

Figure Lengend Snippet: NS1/126-225 inhibits the IFN-β signaling pathway between the level of MAVS and TBK1. (A) 293T cells plated in 24-well plates were transfected with IFN-β-Luc, pRL-TK and empty vector, RIG-I(N), MAVS, TBK1, IKKε, or IRF3(5D) expression plasmids along with a control vector, NS1, or NS1/126-225. Luciferase activity and the transcription of IFN-β were measured at 24 h after transfection. (B) 293T cells plated in 12-well plates were transfected with an empty vector or a vector expressing NS1 or NS1/126-225 along with TBK1 or IRF3(5D), then IFN-β, OASL, PKR, and Mx1 mRNA levels were determined by real-time PCR. The transcript level of each gene was normalized to the expression of β-actin. (C) Production of IFN-β by TBK1 in the presence of NS1/126-225. A549 cells plated in 12-well plates were transfected with an empty vector, NS1, or NS1/126-225 along with TBK1. After 24 h, the amount of IFN-β in the supernatants was measured by ELISA. Western blot analysis of lysates was performed. The data are presented as mean ± SD derived from three repeat experiments. * P < 0.05 or ** P < 0.01, *** P < 0.001 (Student’s t -test).

Article Snippet: Then 100 μl of the cleared medium or the IFN-β standard was used in duplicate for detection of IFN-β using a human IFN-β ELISA Kit (Elabscience, E-EL-H0085c), according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

Journal: Frontiers in Immunology

Article Title: The C-Terminal Effector Domain of Non-Structural Protein 1 of Influenza A Virus Blocks IFN-β Production by Targeting TNF Receptor-Associated Factor 3

doi: 10.3389/fimmu.2017.00779

Figure Lengend Snippet: NS1/126-225 disrupts the formation of MAVS–TRAF3 complex. (A) Schematic diagram of the TRAF3 protein and the following deletion mutants: Flag-TRAF3-ΔTD (AA1 to 346) and Flag-TRAF3-TD (AA347 to 568). (B) Co-IP and western blotting of 293T cells transfected with HA-tagged NS1/126-225 along with a vector expressing the indicated Flag-tagged TRAF3 truncations or full-length TRAF3. An empty vector was used as a negative control. (C) 293T cells were transfected with Flag-TRAF3 and HA-MAVS together with either HA-NS1/126-225 or HA-NS1 and Myc-RIG-I(N) for 36 h. (D) 293T cells were transfected with Flag-TRAF3 and HA-MAVS together with or without Myc-RIG-I(N) for 24 h, then infected with 0.1 MOI HM virus for 12 h. Cell lysates were harvested, then subjected to immunoprecipitation using an anti-Flag antibody, and analyzed by western blotting. (E) 293T cells were transfected with HA-NS1/126-225 as well as Flag-IKKε and HA-MAVS for 24 h. Cell lysates were collected 12 h post-infection with Sev. Co-IP was performed with anti-Flag, and the precipitates were probed with anti-Flag and anti-HA antibodies. (F) A549 cells in 12-well plates were transfected with NS1/126-225, NS1 or an empty vector along with MAVS and TRAF3, together with or without RIG-I(N) for 36 h; NS1/126-225 or NS1 cotransfected with MAVS and IKKε for 24 h, then cells were infected with Sev or mock infected for 12 h. IFN-β concentrations in the supernatants were measured by ELISA. All experiments were performed at least three times. Statistical significance was analyzed with a two-tailed student’s t -test (* P < 0.05 or ** P < 0.01).

Article Snippet: Then 100 μl of the cleared medium or the IFN-β standard was used in duplicate for detection of IFN-β using a human IFN-β ELISA Kit (Elabscience, E-EL-H0085c), according to the manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Negative Control, Infection, Virus, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Two Tailed Test