rabbit anti bcl 6 antibody  (Bioss)

Journal: The Journal of Clinical Investigation

Article Title: c-Rel gain in B cells drives germinal center reactions and autoantibody production

doi: 10.1172/JCI124382

Figure Lengend Snippet: (A) Scheme of the RelTG and GFP-RelTG BAC-transgenic loci. A CAG promoter followed by a loxP-flanked STOP cassette, an N-terminal HA tag or GFP fusion, and a carboxy-terminal FLAG tag were inserted. (B) Representative flow cytometry plots of GCB cells (B220+/CD19+ CD95hiCD38lo). Displayed numbers are median percentages of GCB cells within all B cells (n ≥ 8). Representative flow cytometry histograms of intracellular Bcl6 protein expression. (C) Cell numbers of CD95hiCD38lo GCB cells. Individual data points obtained in 3 or more independent experiments are plotted. Bars and numbers below graphs are median values. **P ≤ 0.01, ***P ≤ 0.001, 1-way ANOVA. (D) Frequencies of light zone (LZ; CXCR4loCD86hi) and dark zone (DZ; CXCR4hiCD86lo) GCB cells and DZ/LZ ratio. Individual data points are plotted. Numbers below graphs and bars are median values for frequencies and geometric means for ratios. *P ≤ 0.05, **P ≤ 0.01, unpaired t test. (E) Cell numbers of CD138+B220lo plasma cells. Individual data points obtained in 6 or more independent experiments are plotted. Bars and numbers below graphs are median values. *P ≤ 0.05, **P ≤ 0.01, unpaired t test. (F and G) Representative flow cytometry plots (F) and pie charts (G) of intracellular Ig isotype staining in plasma cells. Displayed median percentages include data from 8–14 mice analyzed in 5 or more independent experiments. Significant differences with respect to the CD19CreI/+ control genotype are indicated by asterisks adjacent to the respective percentages in the RelTG CD19CreI/+ charts. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired t test. BAC, bacterial artificial chromosome; HygroR, hygromycin B resistance; SPL, spleen; LN, lymph nodes; MLN, mesenteric lymph nodes; PP, Peyer’s patches; BM, bone marrow; ND, not determined. See Supplemental Figures 1–4.

Article Snippet: Sections were stained by incubation at 4°C for 2 days with goat anti–c-Rel (AF2699, R&D Systems) and rabbit anti-Bcl6 antibodies (N-3, Santa Cruz Biotechnology) in dilution buffer (1% BSA and 0.5% Triton X-100 in PBS).

Techniques: Transgenic Assay, FLAG-tag, Flow Cytometry, Expressing, Clinical Proteomics, Staining, Control

Journal: The Journal of Clinical Investigation

Article Title: c-Rel gain in B cells drives germinal center reactions and autoantibody production

doi: 10.1172/JCI124382

Figure Lengend Snippet: (A) CD95hiCD38lo GCB and CD138+B220lo plasma cell numbers 10–12 days after SRBC immunizations obtained from 4 independent experiments. Bars and numbers below graphs are medians. *P ≤ 0.05, ***P ≤ 0.001, paired t test. (B) Areas of splenic GCs quantified based on Bcl6 immunofluorescence in 5 CD19CreI/+ and 5 RelTG CD19CreI/+ mice 10 days after SRBC immunizations (CD19CreI/+, n = 61; RelTG CD19CreI/+, n = 53). Bars and numbers below graphs are means. **P ≤ 0.01, unpaired t test. (C) Percentages of Ig subtypes in splenic plasma cells obtained by intracellular flow cytometry 10–12 days after SRBC immunization. Displayed medians include data from 6 mice from 2 independent experiments. **P ≤ 0.01, paired t test. (D) Individual anti-SRBC IgG1 titers analyzed by flow cytometry 10 days after immunization of 2 independent experiments. Bars and numbers below graphs are medians. (E) NP-CG–immunized mice analyzed after 14 days: Individual percentages of CD95hiCD38lo GCB and IgG+NP+ GCB cells within all splenic B cells are plotted. Bars and numbers below graphs are medians. *P < 0.05, unpaired t test. (F and G) Specific IgG1 serum titers measured by ELISA and calculated by absorbance summation (61) following NP-CG immunizations. Titers for NP2 (high affinity) and NP23 (high and low affinity) (F) and the NP2/NP23 ratio (G) are displayed from 2 independent immunizations. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, multiple t tests. (H) Affinity maturation 14 days after NP-CG immunizations analyzed by sequencing of IgG1 VH186.2 rearrangements in GCB cells for 2 independently immunized cohorts with 2 mice per genotype: 4 CD19CreI/+ mice: immunization I, n = 126 sequences; immunization II, n = 148 sequences; 4 RelTG CD19CreI/+ mice: immunization I, n = 122 sequences; immunization II, n = 117 sequences. The percentage of sequences with the VH186.2 W33L mutation is shown. SPL, spleen; Imm, immunized; ND, not determined. See Supplemental Figures 5 and 6.

Article Snippet: Sections were stained by incubation at 4°C for 2 days with goat anti–c-Rel (AF2699, R&D Systems) and rabbit anti-Bcl6 antibodies (N-3, Santa Cruz Biotechnology) in dilution buffer (1% BSA and 0.5% Triton X-100 in PBS).

Techniques: Clinical Proteomics, Immunofluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Sequencing, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: c-Rel gain in B cells drives germinal center reactions and autoantibody production

doi: 10.1172/JCI124382

Figure Lengend Snippet: (A) Confocal immunofluorescence quantification of nuclear c-Rel intensities of B220+Bcl6– and B220+Bcl6+ cells for exemplary spleen sections from SRBC-immunized RelTG CD19CreI/+ and CD19CreI/+ mice is shown as scatter plots. Histograms depict median nuclear c-Rel intensities for all analyzed mice showing individual data points for each mouse. Green lines, bars, and numbers below graphs are median values (scale bars: 20 μm). *P ≤ 0.05, ****P ≤ 0.0001, unpaired t test. (B) 3D quantification of nuclear c-Rel in splenic B cell nuclei of an exemplary SRBC-immunized RelTG CD19CreI/+ mouse. Insert of a stained section (left; see Supplemental Figure 8C) shows cytoplasmic and nuclear c-Rel staining. Reconstructed surface objects (right) show abundant localization of c-Rel protein (green) in nuclei of Bcl6+ (red, n = 95) versus Bcl6– (white, n = 125) B cells. Graphs below show mean c-Rel 3D intensities in individual nuclei for the depicted insert (left) and for an entire spleen section (right) of B220+Bcl6– mantle zone cells (n = 5980) and B220+Bcl6+ cells (n = 1767); green lines and numbers below graphs depict median values (scale bars: 10 μm). ****P ≤ 0.0001, unpaired t test. (C) Nuclear c-Rel assessed by immunohistochemistry. Graphs display histogram of nuclear c-Rel intensities in B220+Bcl6– and B220+Bcl6+ cells in 3 RelTG CD19CreI/+ and 3 CD19CreI/+ mice (SRBC-immunized) and median nuclear c-Rel intensities for each mouse. Bars and numbers below bars are median values. Exemplary images are shown (scale bars: 50 μm). *P ≤ 0.05, ***P ≤ 0.001, unpaired t test. (D) Frequency distribution of nuclear c-Rel intensities and median nuclear c-Rel intensities in CD20+Bcl6– and CD20+Bcl6+ cells in tonsils of 3 human individuals assessed by immunohistochemistry. Exemplary images are shown (scale bar: 50 μm). **P ≤ 0.01, unpaired t test. See Supplemental Figures 8 and 9.

Article Snippet: Sections were stained by incubation at 4°C for 2 days with goat anti–c-Rel (AF2699, R&D Systems) and rabbit anti-Bcl6 antibodies (N-3, Santa Cruz Biotechnology) in dilution buffer (1% BSA and 0.5% Triton X-100 in PBS).

Techniques: Immunofluorescence, Staining, Immunohistochemistry