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Defective Lymphotoxin-b Receptor-Induced NF-kB Transcriptional Activity in NIK-Deficient Mice
L. Yin, L. Wu, H. Wesche, C.D. Arthur, J.M. White, D.V. Goeddel, R.D. Schreiber
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Supplementary Material
Supplemental Figure 1. Targeting of the mouse NIK gene. (
A) Maps of the NIK targeting vector and the WT and mutant loci. Exon 1 is represented by a black box and the neomycin resistance gene cassette is indicated by an open box. The probe used for Southern blot analysis is shown as a solid line together with the expected hybridization fragment size. The PCR primers used for screening are shown as arrows. The PCR primers for WT NIK alleles were 5´-AGTCCAATTCCATGTTGCTGCTGT-3´ (NIK7) and 5´-TCTGAGATAGGCATATCCCTGGCT-3´ (NIK6). The primers for the disrupted allele were NIK7 and 5´-ATCTTGTTCAATGGCCGATCCCAT-3´ (Neo ATG). (
B) Southern blot analysis of genomic DNA isolated from NIK
+/+, NIK
+/-, and NIK
-/- mice. (
C) Cell lysates of pooled lung, kidney and spleen tissues from one WT or NIK
-/- mouse were immunoprecipitated with antibodies specific for NIK (top panel, mAb against human NIK, Tularik) or NEMO (bottom panel, Santa Cruz) and immunoblotted with anti-NIK or a mixture of anti-IKK
a and anti-IKK
b respectively.
Medium version | Full size version
Supplemental Figure 2. Abnormal immune system development in NIK-/- mice. (A) Lack of inguinal LN in NIK-/- mice. NIK-/-and WT mice were injected in the hind footpads with 50 ml India ink and inguinal LN were inspected 30 min after injection. Black staining reveals the presence of LN in WT but not NIK-/- mice. (B and C) Abnormal lymphoid architecture in NIK-/- mice. H&E staining of thymus (TH) (B) or spleen (SP) (C) sections from NIK-/- or WT mice (magnification: 10×). (D) Abnormal antibody responses in NIK-/- mice. Serum Ig levels of unimmunized (U) or ovalbumin immunized (I) WT mice and NIK-/- mice were measured by ELISA. For immunization, mice were injected intraperitoneally (i.p) with 100 mg ovalbumin emulsified in complete Freunds adjuvant followed by an i.p boost with the same amount of antigen with incomplete Freunds adjuvant 2 weeks later. Serum was collected 10 days after boosting. Three mice were used for each group. The experiment was repeated one additional time with an independent set of mice.
Medium version | Full size version
| Supplemental Table 1. Hematopoiesis in WT and NIK-/- mice.*
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| Thymus | Cellularity (×10-8) | CD4+CD8- (%) | CD4-CD8+ (%) | CD4+CD8+ (%) |
| NIK+/+ | 1.68 ± 0.05 (3) | 8.93 ± 0.49 (4) | 3.88 ± 0.88 (4) | 83.31+1.84 (4) |
| NIK-/- | 1.43 ± 0.28 (5) | 16.26 ± 1.86 (6) | 3.73 ± 0.84 (6) | 77.82+2.18 (6) |
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| Spleen | Cellularity (×10-8) | CD4+T cells (%) | CD8+T cells (%) | B220+IgM+ (%) | Cd11b+ monocytes (%) |
| NIK+/+ | 0.99 ± 0.19 (6) | 19.35 ± 2.34 (8) | 11.78 ± 3.50 (8) | 40.66 ± 6.80 (7) | 7.70 ± 2.74 (7) |
| NIK-/- | 0.91 ± 0.19 (6) | 22.28 ± 4.78 (8) | 27.53 ± 11.93 (8) | 16.56 ± 3.38 (7) | 8.81 ± 2.24 (6) |
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| Bone Marrow | B220-CD43+ (%) | B220+CD43+ (%) | B220+CD25+ (%) | B220+CD43- (%) | B220+IgM+ (%) |
| NIK+/+ | 32.90 ± 8.03 (6) | 10.27 ± 1.82 (6) | 31.0 ± 5.2 (3) | 47.40 ± 8.01 (6) | 20.87 ± 2.62 (4) |
| NIK-/- | 21.10 ± 5.39 (7) | 11.74 ± 3.49 (7) | 51.53 ± 1.75 (3) | 61.06 ± 12.21 (7) | 18.92 ± 2.45 (5) |
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| *Cells from thymi, spleens and bone marrow of WT and NIK-/- mice were stained with antibodies against the indicated surface markers and analyzed by flow cytometry. Numbers indicate mean percentages ± SD of positive cells per total cells. The number of mice analyzed is shown in the parentheses. Bone marrow cells were gated on lymphocytes only. |
