Journal: Cell reports

Article Title: The miR-17∼92 miRNAs promote plasma cell differentiation by suppressing SOCS3-mediated NIK degradation.

doi: 10.1016/j.celrep.2023.112968

Figure Lengend Snippet: Figure 6. SOCS3 promotes NIK ubiquitination and degradation (A) Cell lysates from WT iGCB cells cultured with 40LB plus IL-21 for 24 h were immunoprecipitated (IP) with anti-SOCS3 or IgG and analyzed by immunoblotting (IB) of SOCS3 and NIK. (B) Immunoblot analysis of NIK and GAPDH in WT and TKO iGCB cells cultured with 40LB cells plus IL-21 and treated with DMSO or MG132 for indicated lengths of time. (C) HEK293T cells were transfected with indicated vectors and treated with MG132. Cell lysates were immunoprecipitated with anti-FLAG (NIK) antibody. Ubiquitinated and total NIK was detected by immunoblot analysis of anti-HA and anti-FLAG antibodies, respectively (upper). Immunoblot analysis of SOCS3, FLAG (NIK), HA (Ub), and GAPDH in whole-cell lysates (WCL) (lower). (D) Flow-cytometry analysis of iPCs among WT and TKO iGCB cells transduced with empty retroviruses (Vector) or retroviruses encoding NIK, IRF4, and Blimp1 (upper). Summary of the percentage of iPCs (lower). Small horizontal lines indicate the means (±SEM). *p < 0.05, ***p < 0.001. Data are representative of three independent experiments. See also Figure S9.

Article Snippet: Anti-IgG1 or anti-NP IgG1 spots were detected by biotin-conjugated anti-mouse IgG1 antibody (Southern Biotechnology, 1070-08) in combination with Av-HRP and AEC substrate (Vector Laboratories, A-2004& SK-4200).

Techniques: Ubiquitin Proteomics, Cell Culture, Immunoprecipitation, Western Blot, Transfection, Flow Cytometry, Transduction, Plasmid Preparation

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: Following an incubation period of 1-6 weeks, HFRS symptoms manifest as five distinct phases: febrile, hypotensive, oliguric, polyuric, and convalescent. The average time for which each stage lasts is shown, in addition to the symptoms associated with each phase. Serum creatinine, a measure of kidney injury, is displayed, as is urine output as a marker of kidney function. IgM and IgG titers are also shown. The four time points at which patient sera were sampled are also shown in relation to each disease phase. Time point 1 (T1) was sampled six days post-symptom onset (PSO), range: 2-11 days PSO). Time point 2 (T2) was sampled 12 days PSO (range: 8-20 days PSO). Time point 3 (T3) was sampled 28 PSO (range: 21-64 days PSO). Time point 4 (T4) was sampled 197 days PSO (range: 180-256 days PSO). Adapted from Avsic-Zupanc et al. . Created in BioRender. Clark, J. (2025) https://BioRender.com/p36u208

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Incubation, Marker

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: ELISAs were carried out using patient sera and plates coated with ultracentrifuge purified rVSV expressing the GnGc of A) PUUV, B) SEOV, C) DOBV, D) HTNV, E) SNV, or F) ANDV. Wild-type VSV G) was used as a negative control. IgG titers are shown as area under the curve (AUC) with the geometric mean and geometric standard deviation denoted by error bars. 11 age and sex matched healthy control sera were included as negative controls and an in-house, anti-PUUV monoclonal antibody was used as a positive control. AUC values were log10 transformed and statistical significance was interrogated via ordinary one-way ANOVA using a Tukey’s multiple comparisons test. Only comparisons with p ≤ 0.05 are shown. Limits of detection (LOD) were calculated for each antigen using the average + 3x standard deviations of the AUC of the negative control sera and are shown as dotted lines on each graph. The percentage of serum samples with LODs above the limit of detection are shown as pie charts, with the percentage of positive samples displayed in green. The n for each time point is displayed below each pie chart.

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Purification, Expressing, Negative Control, Standard Deviation, Control, Positive Control, Transformation Assay

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: ELISAs were carried out using patient sera and plates coated with recombinant truncated, Gn of A) PUUV and B) SEOV. IgG titers are shown as area under the curve (AUC) with the geometric mean and geometric standard deviation denoted by error bars. 11 age and sex matched healthy control sera were included as negative controls and an in-house, anti-PUUV monoclonal antibody was used as a positive control. AUC values were log10 transformed and statistical significance was interrogated via ordinary one-way ANOVA using a Tukey’s multiple comparisons test. Only comparisons with p ≤ 0.05 are shown. Limits of detection (LOD) were calculated for each antigen using the average + 3x standard deviations of the AUC of the negative control sera and are shown as dotted lines on each graph. The percentage of serum samples with LODs above the limit of detection are shown as pie charts, with the percentage of positive samples displayed in green. The n for each time point is displayed below each pie chart.

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Recombinant, Standard Deviation, Control, Positive Control, Transformation Assay, Negative Control

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: ELISAs were carried out using patient sera to investigate A) anti-PUUV IgM, B) anti-PUUV IgA, C) anti-SEOV IgM, and D) anti-SEOV IgA. IgG subtype ELISAs were performed to investigate the quantities of anti-PUUV E) IgG1, F) IgG2, G) IgG3, or H) IgG4. I) A schematic of the luciferase-based reporter assay, which was utilized to characterize the effector activity promoted by patient sera. RVSV-PUUV infected Huh.75 cells were incubated with patient sera and reporter cells that express the FcγRIIIa receptor, coupled to a gene expression pathway that results in the production of luciferase. J ) The FcγRIIIa activity of the sera re shown as AUCs, with the mean and standard deviation denoted by error bars. 11 age and sex matched healthy control sera were included as negative controls, and an in-house, anti-PUUV monoclonal antibody was used as a positive control. AUC values were log10 transformed and statistical significance was interrogated via ordinary one-way ANOVA using a Tukey’s multiple comparisons test. Only comparisons with p ≤ 0.05 are shown. Limits of detection (LOD) are shown as dotted lines on each graph. The percentage of serum samples with LODs above the limit of detection are shown as pie charts with the percentage of positive samples displayed in green. The n for each time point is displayed below each pie chart.

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Luciferase, Reporter Assay, Activity Assay, Infection, Incubation, Gene Expression, Standard Deviation, Control, Positive Control, Transformation Assay