Journal: eLife

Article Title: Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node

doi: 10.7554/eLife.62781

Figure Lengend Snippet: ( a ) Bone marrow- derived dendritic cells (BMDCs) were grown for 7 days and cultured in GM-CSF. After 7 days, 5 mg of either pDNA, psDNA, ova-pDNA, or ova-psDNA were added to the culture media, and 1, 3, or 7 days after addition, media was removed and qPCR was performed on using primers against the DNA added. 3–5 wells were evaluated per group on 2–3 independent occasions. ( b ) As in ( a ) except with murine lymph node lymphatic endothelial cells. ( c ) As in Figure 1h, I , BMDCs were treated with ovalbumin (ova) (5 μg) ± polyI:C and anti-CD40 (20 μg each) or ova-psDNA ± polyI:C and anti-CD40 overnight and then co-cultured with Carboxyfluorescein Succinimidyl Ester (CFSE)-labeled OT1 T cells for 3 days before evaluating CFSE dilution by flow cytometry. Experiments were performed three times with 3–5 wells per sample with similar results.

Article Snippet: Antibody , Anti-mouse CD40 (Rat monoclonal) , BioXcell , Cat#BE0016-2 , .

Techniques: Derivative Assay, Cell Culture, Labeling, Flow Cytometry

Journal: eLife

Article Title: Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node

doi: 10.7554/eLife.62781

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse CD40 (Rat monoclonal) , BioXcell , Cat#BE0016-2 , .

Techniques: Recombinant

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: p190RhoGEF protein levels and changes after CD40 stimulation of B cells of various maturational stages. (A) Mouse splenic B cells (1 × 10 6 /ml) and the B cell lines WEHI 231, BAL17, CH12.LX (5 × 10 5 /ml) were stimulated with isotype control Ig or anti-CD40 at a concentration of 1 µg/ml for 48 h. (B) HEK 293T cells were transfected with plasmids carrying an empty vector (EV), WT, or DN p190RhoGEF (HA). (C) WEHI 231 B cells were stably transfected with MIGR1 (EV), WT, or DN p190RhoGEF. Lysates were prepared from these cells, subjected to a SDS-PAGE (6% or 8% gels), transferred to nitrocellulose, and probed with an Ab specific for p190RhoGEF (A-C) or HA (B). Equal loading of the protein samples was demonstrated by anti-β actin IB in (A). (D, E) Mouse splenic B cells (1 × 10 6 /ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 µg/ml. After 48 h of stimulation, cells were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD23, and IgD. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing IgD versus CD23 positive cells. For the cell populations in R1, R2, and R3 as indicated in (D), the population of cells (%) and the expression level of p190RhoGEF as mean fluorescence intensity (MFI) in the absence (control) and presence of CD40 stimulation is presented in (E). The results shown are representative of three independent experiments.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Control, Concentration Assay, Transfection, Plasmid Preparation, Stable Transfection, SDS Page, Staining, Fluorescence, Flow Cytometry, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Expression of p190RhoGEF, CD138, and B220 in B cells at various maturational stages. (A, B) Mouse splenic B cells (1 × 10 6 /ml) were stimulated with control Ig or anti-CD40 at a concentration of 1 µg/ml. After 72 h of stimulation, cells were fixed, permeabilized, and stained for endogenous p190RhoGEF, B220, and CD138. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results from flow cytometry are presented as a dot plot, showing the B220 positive cells versus the CD138 positive cells. The data shown are representative of three independent experiments. For the cell populations indicated in (A), expression of p190RhoGEF is presented as MFI in (B). Data are presented as the mean ( n = 4) ± SD. (C, D) The B cell lines, BAL17, CH12.LX, and NS-1 were fixed, permeabilized, and stained for endogenous p190RhoGEF, CD138, and B220. The results were obtained as described above. For the cell populations indicated in (C), expression of p190RhoGEF is presented as MFI in (D). Data are presented as the mean ( n = 3) ± SD.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Control, Concentration Assay, Staining, Fluorescence, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Expression changes of transcriptional regulators after CD40 stimulation and in response to p190RhoGEF and RhoA down-regulation in B cells at various maturational stages. (A) cDNA was prepared from B cells that were either untreated (control) or stimulated for 24 h with anti-CD40 (1 µg/mL). (B) cDNA was prepared from BAL17 and NS-1 cells that were stably transfected with MIGR1 (EV), p190RhoGEF (Y1003A) or RhoA (T19N). The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Control, Stable Transfection, Transfection

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Direct effects of manipulating the activity of p190RhoGEF and RhoA on B cell maturation and downstream transcriptional regulators. WEHI 231 B cells were stably transfected with MIGR1 (EV), WT p190RhoGEF, DN p190RhoGEF (Y1003A), CA RhoA (Q63L), DN RhoA (T19N), or a combination of WT p190RhoGEF and DN RhoA (T19N). (A) cDNA was prepared from these stably transfected cells. The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, Bcl-6, AID, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH. (B) These stably transfected cells were removed from the cultures and then washed, counted, and recultured in fresh medium for 24 h at 1 × 10 6 cells/ml. Some EV-transfected cells were also incubated with a medium alone (black bars) or with a combination of CD40 and IL4 as positive controls (white bars). Supernatants were harvested and serially diluted, and secreted Ig was measured by anti-IgM and anti-IgG ELISA as described in "Methods." Values of the optical densities at 490 nm in triplicate supernatant samples were collected in each independent experiment. Data are presented as the mean of three independent experiments ( n = 3) ± SD, * P < 0.05, ** P < 0.01, compared with cells transfected with EV. (C, D) These stably transfected cells were fixed, stained with anti-CD138 or anti-CXCR4, and analyzed by standard flow cytometry. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown).The bar graphs show the expression of CD138 (C) or CXCR4 (D), as indicated by MFI. Data are presented as the mean of three independent experiments ( n = 3) ± SEM, ** P < 0.01, *** P < 0.001, compared with cells transfected with EV.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Activity Assay, Stable Transfection, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Fluorescence, Control, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

doi: 10.3858/emm.2012.44.2.009

Figure Lengend Snippet: Effects of the overexpressed DN forms of p190RhoGEF or RhoA on the expression of CD138, CXCR4, and transcriptional regulators after CD40 stimulation. WEHI 231 B cells were stably transfected with either MIGR1 (EV), a plasmid encoding DN p190RhoGEF (Y1003A), or a plasmid encoding DN RhoA (T19N). (A, B) After CD40 stimulation for 48 h, cells were fixed, stained with anti-CD138 or anti-CXCR4, and measured by flow cytometry. The fluorescence intensities of cells stained with an isotype control Ab or antiserum were all corrected (data not shown). The results show the expression intensities of CD138 (A) or CXCR4 (B). Data are presented as the mean of three independent experiments ( n = 3) ± SD. (C) cDNA was prepared from these stably transfected cells, after being stimulated for 48 h with anti-CD40 Ab (1 µg/ml). The PCR products for p190RhoGEF, RhoA, Blimp-1, XBP-1, IRF-4, and GAPDH were separated as described in "Methods." The data shown are representative of three independent experiments. Values indicate normalization for GAPDH.

Article Snippet: The rat anti-mouse CD40 mAb (clone 1C10) from R&D Systems, Inc. (Minneapolis, MN) and the hamster anti-mouse CD40 mAb (HM40-3) from BD PharMingen (San Diego, CA) were used to stimulate B cells.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: Uncoupling Splicing From Transcription Using Antisense Oligonucleotides Reveals a Dual Role for I Exon Donor Splice Sites in Antibody Class Switching

doi: 10.3389/fimmu.2020.00780

Figure Lengend Snippet: Specific IgG1 class switching inhibition in B cells treated by Iγ1 exon dss ASO. Splenic B cells were isolated from C57BL / 6 mice, stimulated with anti-CD40 + IL4 and treated with 2 μM Iγ1 dss ASO (ASO) or an irrelevant ASO (control) for 2 days (B,E) or 4 days (C,D,F,G) . (A) Schematic representation of unspliced ε GLT. Iε, ε I exon; Sε, ε switch region; CH1ε, ε constant exon 1; dss, donor splice site. (B,E) Unspliced γ1 (B) and ε (E) GLT expression monitored as described in . Iε-for-Q and SεU-rev-Q primers, described in schema A , were used for ε GLT expression determination. (C,F) Post-switch Iμ-Cγ1 and Iμ-Cε mRNA expressions monitored as described in . (D,G) Quantification of IgG1 and IgE in culture supernatants by ELISA. (B–G) Data are means ± SEM of two independent experiments, n = 3–4 for each group. Unpaired two-tailed Student's t test was used to determine significance. ns: non-significant, ** P <0.01, *** P <0.001.

Article Snippet: Splenic B cells were stimulated with either 1 or 5 μg/ml lipopolysaccharide (LPS) (LPS-EB Ultrapure, InvivoGen), 1 μg/ml LPS + 20 ng/ml interleukin 4 (IL4) (recombinant murine IL-4, PeproTech), or 5 μg/ml anti-CD40 [mouse CD40/TNFRSF5 MAb (Clone 1C10), Bio-Techne, USA] + 40 ng/ml IL4.

Techniques: Inhibition, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test