(A) Photographs of inguinal LNs in situ from WT and Nfkbia^NES/NES littermates (top). LNs were counted in WT (n = 35) and Nfkbia^NES/NES (n = 35) mice and displayed graphically (bottom).
(B) Inguinal LNs in WT and Nfkbia^NES/NES mice were analyzed by hematoxylin and eosin (H&E) stain and immunostaining with anti-B220.
(C) Inguinal LN volume was derived by the formula width² × length/2 of histologic sections from wild-type (n = 32) and Nfkbia^NES/NES (n = 11) mice and displayed graphically. The difference is statistically significant (*p < 0.001, unpaired t test).
(D) PPs were counted from WT (n = 6) and Nfkbia^NES/NES (n = 7) mice and displayed graphically.
(E) Aggregate size of PPs were calculated as in (C) from samples in (D). Data are mean ± SD, *p < 0.001 versus WT.
(F) Spleens from WT and Nfkbia^NES/NES mice were analyzed by H&E stain and immunostaining with anti-B220, 4× objective.
(G) Immunofluorescent histochemical analysis of the spleens from WT or Nfkbia^NES/NES mice were analyzed with anti-MOMA-1 and anti-IgM. MZ B cell layer is external to the ring of metallophilic macrophages.
(H) Thymii from WT or Nfkbia^NES/NES mice were analyzed by H&E stain, 4× objective.

Comparisons between WT and Nfkbia^NES/NES mice are shown in each panel.
(A) Splenocytes were stained with anti-B220 and anti-Thy1.2. Percentages above each box indicate cells in the gated lymphoid population.
(B) The numbers of total splenocytes and total splenic B and T cells are displayed graphically.
(C) Thymocytes from WT and Nfkbia^NES/NES mice were stained with anti-CD4 and anti-CD8. Percentages indicate cells in the gated lymphoid population (C and E–G).
(D) The numbers of total thymocytes and DN, DP, CD4⁺, and CD8⁺ T cells in the thymus of WT and Nfkbia^NES/NES mice are displayed graphically.
(E) Splenocytes were stained with anti-B220, anti-IgM, and anti-IgD. For cells gated on B220⁺, T1 (IgM^hiIgD⁻), T2 (IgM^hiIgD⁺), and FO (IgM^loIgD⁺) B cells are shown.
(F) Splenocytes were stained with anti-IgM, anti-CD21, and anti-CD23. For cells gated on CD23⁺, T2 (CD21^hiIgM^hi) and FO (CD21^intIgM^lo) B cells are shown. For cells gated on CD23⁻, T1 (CD21^loIgM^hi) and MZ (CD21^hiIgM^hi) B cells are shown.
(G) Splenocytes were stained with anti-B220, anti-CD21, and anti-CD23. For cells gated on B220⁺, MZ B cells (CD21^hiCD23^lo) are shown.
(H) Top: Splenocytes were stained with anti-B220, anti-CD1d, and anti-CD9. For cells gated on B220⁺, MZ (CD1d⁺CD9⁺) cells are shown. Bottom: Splenocytes were stained with anti-IgM, anti-CD1d, and anti-CD23. For gated lymphoid cells, T2 (CD1d⁺CD23⁺) and MZ (CD1d⁺CD23⁻) cells are shown.
(I) The numbers of T1, T2, FO, and MZ B cells obtained from (F) are displayed graphically.
(J) Serum Ig amounts were determined by ELISA. The mean value and standard deviation of the serum Ig amounts were calculated.
Data shown are representative of or obtained from 7 (A–G, I), 2 (H), or 10 (J) mice of each genotype.

Sublethally irradiated Jak3^−/− mice were transplanted with BM from WT (Jak3^−/− + Nfkbia^+/+) or Nfkbia^NES/NES (Jak3^−/− + Nfkbia^NES/NES) mice. Irradiated Jak3^−/− mice without BM transplantation were used as a negative control.
(A) BM from the recipient mice were stained with anti-B220 and anti-IgM. Percentages here and below indicate cells in the gated lymphoid population.
(B) Splenocytes from the recipients were stained with anti-B220 and anti-Thy1.2.
(C) Splenocytes from the recipients were stained with anti-IgM, anti-CD21, and anti-CD23. In cells gated on CD23⁺, T2 and FO B cells are shown. In cells gated on CD23⁻, T1 and MZ B cells are shown.
(D) Splenocytes from the recipients were stained with anti-B220, anti-CD21, and anti-CD23. In cells gated on B220⁺, MZ B cells are shown.
Data are representative of five recipients transplanted with each genotype of BM. Controls in (C) and (D) are not shown due to very low percentages.

(A) Total cell lysates of unstimulated splenic AA4.1⁻ mature B cells from WT and Nfkbia^NES/NES mice were analyzed by a supershift assay to detect different NF-κB family members.
(B and C) Splenic AA4.1⁻ mature B cells of WT and Nfkbia^NES/NES mice were stimulated with anti-IgM (10 µg/mL, B) and LPS (10 µg/mL, C) for the indicated times. Total cell extracts were made and NF-κB activity was measured by EMSA with an Igκ-κB probe. Variable increases in p50 homodimer binding seen in (B) are probably due to enhanced detection of p50 homodimer that has a lower affinity to the Igκ-κB site used because of the lack of competition by high-affinity heterodimer complexes for a limited amount of probe used. A short exposure time is shown in (C) to highlight the difference of induced activity (the basal activity difference is thus less evident because of underexposure).
(D and E) Samples in (B) and (C) were analyzed by immunoblotting with anti-IκBα, -IκBβ, and -actin.
(F) The IKK complex was immunoprecipitated from B cells stimulated with LPS (10 µg/mL for 30 min) and an IKK kinase assay was performed with GST-IκBα(1–66aa) as substrate.
(G) Splenic AA4.1⁻ mature B cells were fixed and stained with anti-IκBα and anti-cRel and counterstained with Hoechst dye.
(H) Percentages ± 1 SD of pronounced nuclear staining for each protein as in (G) were derived from triplicate counts of random 200 cells. **paired t test determined a statistically significant difference p < 0.001.
(I) Quantitation of nuclear and cytoplasmic IκBα and cRel in splenic AA4.1⁻ mature B cells. Images of stained cells as in (G) were analyzed by Image J software. Mean intensity of staining in the nuclear and cytoplasmic compartments of 10 random cells stained with each antibody, normalized to background intensity, and expressed as a ratio of mean nuclear intensity/mean cytoplasmic intensity (N/C) are shown as columns with 1SD bars. Paired t test determined a statistically significant difference in N/C ratio of IκBα and cRel between WT and mutant cells, **p < 0.001.
(J) Lysates obtained from splenic AA4.1⁻ mature B cells of WT and Nfkbia^NES/NES mice were incubated with anti-cRel and the precipitates were analyzed by immunoblotting with anti-cRel, -IκBα, and -IκBβ (left). Inputs were blotted with the same antibodies and an anti-tubulin loading control (right).

(A) Splenic AA4.1⁻ mature B cells were purified from Nfkbia^+/+, Nfkbia^+/NES, and Nfkbia^NES/NES mice and total cell extracts were made. The expression of NF-κB and IκB family members were analyzed by immunoblotting.
(B) Total RNA from splenic AA4.1⁻ mature B cells of WT and Nfkbia^NES/NES mice were analyzed by an RT² Profiler Mouse NF-κB Signaling Pathway PCR Array (SA Biosciences) and the statistical analysis was done according to the manufacturer’s instructions.
(C) Splenic AA4.1⁻ mature B cells of WT and Nfkbia^NES/NES mice were treated with cycloheximide (25 µg/ml) for indicated times or with LPS alone (10 µg/ml, 1 hr) and total cell lysates were analyzed for IκBα and IκBβ degradation by immunoblotting. The relative degradation as measured by laser scanning of each band followed by NIH ImageJ analysis normalized to actin loading control showed that the rate of mutant IκBα degradation was 2-fold slower than that of the WT protein.
(D) Splenic AA4.1⁻ mature B cells purified from WT and Nfkbia^NES/NES mice were treated with BAFF (500 ng/mL) for 24 hr. The processing of p100 to p52 was detected by immunoblot with an anti-p52.
(E) EMSA analysis was performed with samples in (D) via an Igκ-κB probe.
(F) MEF cells generated from Nfkbia^NES/NES and WT mice were treated with anti-LTβR (0.5 µg/ml) for the indicated times (hr). The processing of p100 was detected by immunoblot with anti-p52 along with antibodies to other NF-κB and IκB family members.
(G) EMSA analysis of extracts from (F) was performed as in (E).

(A and B) Splenic immature (AA4.1⁺) and mature (AA4.1⁻) B cells purified from WT and Nfkbia^NES/NES mice were stimulated with anti-IgM with or without IL-4 or with LPS. Proliferation was assessed by [³H]thymidine incorporation. Data are representative of at least four independent experiments.
(C and D) Splenic immature (AA4.1⁺) and mature (AA4.1⁻) B cells were purified from WT and Nfkbia^NES/NES mice and then stimulated with anti-IgM plus IL-4 or with LPS. Subsequently, cells were collected, stained with propidium iodide, and analyzed for cell cycle profile by flow cytometry. The percentages of cells in subG₀, G₀–G₁, and S-G₂-M are indicated.
(E and F) Splenic mature (AA4.1⁻) B cells purified from WT and Nfkbia^NES/NES mice were stimulated with IgM antibody or with LPS. RNA expression amounts of Bcl2l1 (encoding Bcl-X_L) and Myc were determined with real-time reverse transcription PCR. Data were done in triplicate from three and two mice each for Bcl2l1 and Myc, respectively.
(G) Spenic and thymic T cells purified from WT and Nfkbia^NES/NES mice were stimulated with indicated agonists and analyzed as in (A).