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:

Article Title: Melanoma inhibitor of apoptosis protein (ML-IAP) is a target for immune-mediated tumor destruction

doi: 10.1073/pnas.0530311100

Figure Lengend Snippet: Vaccination-augmented anti-ML-IAP antibody titers and induced isotype switching. (A) Pan-IgG. (B) IgG4. The ELISA was performed with recombinant GST-ML-IAP protein and K030 sera diluted 1:10,000 for the pan-IgG determinations and 1:100 for the IgG4 measurements. Small arrows denote vaccinations. The peak pan-IgG and IgG4 levels were 4-fold and 10-fold higher than the respective values at study entry. K030 sera also recognized GST-ML-IAP in Western analysis. Reactivity to Candida antigens was not consistently affected by therapy (data not shown).

Article Snippet: A polyclonal goat anti-human pan IgG (Jackson ImmunoResearch, 1:1,000 dilution), monoclonal mouse anti-IgG1 (Zymed, 1:500 dilution), or monoclonal mouse anti-IgG4 (Southern Biotechnology Associates, 1:500 dilution) conjugated to alkaline phosphatase in 2% NFDM/PBS was added at room temperature for 1 h, and the plate was developed with p -nitrophenyl phosphate (Sigma).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot

Journal:

Article Title: Myeloid Cell Function in MRP-14 (S100A9) Null Mice

doi: 10.1128/MCB.23.7.2564-2576.2003

Figure Lengend Snippet: MRP-14 protein is absent in MRP-14−/− monocytes and neutrophils. (A) Myeloid cells were identified by flow cytometry by staining MRP-14+/+ and MRP-14−/− bone marrow cells with MAb 7/4. (B) The MAb 7/4-positive myeloid cells (R1) from MRP-14+/+ and MRP-14−/− mice were intracellularly stained with either MAb 2B10, specific for MRP-14 (shaded histogram), or an IgG2a isotype control MAb (open histogram). (C) In MRP-14+/+ mice, two different MRP-14-positive populations (R2 and R3) can be seen that are absent in MRP-14−/− myeloid cells. These populations correspond to monocytes (R2) and neutrophils (R3), based on their differential staining with MAb Gr-1. Open histogram represents the Gr-1-negative, 7/4-negative cells. Inset numbers represent the geometric mean fluorescence of three Gr-1-positive populations.

Article Snippet: MRP-14 was detected with MAb 2B10 (2 μg/ml) and compared to control IgG2a, followed by goat anti-rat immunoglobulin-horseradish peroxidase conjugate (1:500, Southern Biotechnology).

Techniques: Flow Cytometry, Staining, Fluorescence