soluble recombinant murine cd40l  (PeproTech)

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A, B) Mean±SEM levels of the indicated cell surface proteins on control and WNK1-deficient B cells stimulated with anti-IgM (A) or CD40L (B) for the indicated times. Data shown as MFI normalized to the maximum response of control B cells (set to 100). (C) Mean±SEM concentrations of the indicated cytokines in medium from cultures of control or WNK1-deficient B cells that were unstimulated or stimulated with anti-IgM or CD40L for 72 h. (D and E) Immunoblot analysis (top) of cell lysates from control or WNK1-deficient B cells stimulated with anti-IgM (D) or CD40L (E) for the indicated times, probed with antibodies to MYC or α-TUBULIN. Graphs (below) show mean±SEM levels of MYC normalized to the abundance of α-TUBULIN in each lane. (F) Immunoblot analysis (top) of cell lysates from wild-type mouse B cells stimulated with LPS for the indicated times, probed with antibodies to p-OXSR1 and α-TUBULIN. Graph shows mean±SEM levels of p-OXSR1 normalized to the abundance of α-TUBULIN in each lane. (G) Top; immunoblots of total cell lysates from wild-type mouse B cells treated with vehicle (DMSO), a PI3K inhibitor (PI-103) or an AKT inhibitor (MK2206) and stimulated for the indicated times with CD40L, probed with antibodies to p-OXSR1, p-AKT, p-PRAS40 or ERK2. Below; graphs of mean±SEM abundance of p-OXSR1, p-AKT and p-PRAS40 in the lanes above, normalized to ERK2. Mann-Whitney test; * 0.01 < P < 0.05, ** 0.001 < P < 0.01, **** P < 0.0001. Sample sizes: 5-6 (A), 6 (B, D, E, G), 9-15 (C), 5 (F). Data are pooled from 2 (A, B, D, E) or 3 (C, F) independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A, B) B cells of the indicated genotypes labelled with Cell Proliferation Dye (CPD) were cultured for 72 h in the presence of anti-IgM, CD40L or LPS. (A) Representative histograms of CPD fluorescence measured by flow cytometry; cell division results in dye dilution. (B) Mean±SEM percentage of B cells that have divided at least once after 72 h stimulation with either anti-IgM, CD40L or LPS. (C) Mean±SEM number of live control or WNK1-deficient B cells after 72 h culture with the indicated stimuli. (D) Mean±SEM number of cells after 72 h culture if there had been no division. (E) Mean±SEM percentage of Ki-67 + B cells after stimulation with anti-IgM or CD40L for the indicated times. (F-K) CPD-labelled B cells of the indicated genotypes labelled with CPD were cultured for 72 h in the presence of anti-IgM or CD40L. (F, H, J) Histograms of CPD fluorescence. (G, I, K) Mean±SEM percentage of B cells that have divided at least once after 72 h in response to the indicated stimuli. (L-N) Top; immunoblots of total cell lysates from mouse B cells stimulated for the indicated times with CD40L using WNK1-deficient or control B cells (L), B cells expressing kinase-inactive WNK1-D368A or control B cells (M), or wild-type B cells treated with vehicle (DMSO), or an inhibitor of WNK-family kinases (WNK463) (N), probed with antibodies to p-OXSR1 or α-TUBULIN. Below; graphs of mean±SEM abundance of p-OXSR1 in the lanes above, normalized to α-TUBULIN. Mann-Whitney test (B, C, D, G, I, K-N), two-way ANOVA (E); * 0.01 < P < 0.05, ** 0.001 < P < 0.01, *** 0.0001 < P < 0.001, **** P < 0.0001. Sample sizes: 5 WNK1-deficient, 6 control (B, D), 9 WNK1-deficient, 15 control (C), 7 WNK1-deficient, 6 control (E), 4 WNK1-D368A, 6 control (G), 6 (I), 7 (K), 5 (L-N). Data pooled from two (B, D, E, G, I, K-N) or three (C) independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Cell Culture, Fluorescence, Flow Cytometry, Western Blot, Expressing, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MiR-210 is induced by Oct-2, regulates B-cells and inhibits autoantibody production 1

doi: 10.4049/jimmunol.1301289

Figure Lengend Snippet: A. B-cells from 8-12 wk old mice of various murine strains (top row) were stimulated with αIgM + CD40L + IL-4 or LPS for 48 hours, and their miRNA profiles compared to resting controls. Each row corresponds to a miRNA gene, and each column to an individual mouse. Red indicates increased, and blue reduced, expression. MiRNAs are ranked according to the degree of differential expression across samples. MiR-210 expression, which is up-regulated upon activation with both types of stimuli, is boxed in black.

Article Snippet: Cell stimulation and transfection For miRNA expression profiling studies, cells were cultured at 2 × 10 6 cells/ml and stimulated with either goat anti–mouse IgM μ-chain specific F(ab′)2 (10 μg/ml: Jackson ImmunoResearch Laboratories) with recombinant murine CD40L (1 μg /ml: PeproTech) and IL-4 (20 ng/ml: PeproTech), or LPS from E.coli (10 μg /ml, Sigma).

Techniques: Expressing, Quantitative Proteomics, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MiR-210 is induced by Oct-2, regulates B-cells and inhibits autoantibody production 1

doi: 10.4049/jimmunol.1301289

Figure Lengend Snippet: A. Cell cycle analysis with propidium iodide of splenic B2 cells stimulated with αIgM + CD40L + IL-4 for 48 hours. Representative FACS plots are shown in the top panel and the percentage of cells in each phase of the cell cycle is quantified in the bottom panel. The average of three mice in each group ± SEM is shown.

Article Snippet: Cell stimulation and transfection For miRNA expression profiling studies, cells were cultured at 2 × 10 6 cells/ml and stimulated with either goat anti–mouse IgM μ-chain specific F(ab′)2 (10 μg/ml: Jackson ImmunoResearch Laboratories) with recombinant murine CD40L (1 μg /ml: PeproTech) and IL-4 (20 ng/ml: PeproTech), or LPS from E.coli (10 μg /ml, Sigma).

Techniques: Cell Cycle Assay