Changes in sCLU protein level in spleen and MLN of WT mice at day 8 after immunization with SRBC.

<p>Western blot of total tissue homogenates shows an increase in sCLU amount in spleen but not MLN. Data is representative of 2 independent experiments.</p

Clusterin expression in activated MEF.

<p>(A) MEF were incubated with either Reh cells bearing surface LT (“LT+”) or Jurkat cells not expressing LT on their surface (“LT−”) for indicated time periods, and clusterin mRNA was measured by real-time RT-PCR. Data was normalized to mouse β-actin. (B) Physical interaction of MEF with lymphoid cells in culture. Data is represented as mean±SD.</p

Relative mRNA levels of known and potential LTβR target genes in various mouse tissues.

<p>Real-time PCR data on mRNA levels in various tissues from wild type, TNFR1-KO, and LTβR-KO mice of selected genes down-regulated in LTβR-KO splenic stroma. Data was normalized to GAPDH, which expression level was taken as 100%. Note high expression of clusterin in wild type spleen and its dramatic reduction in spleen upon LTβR knockout. Data is represented as mean±SD. * – Difference from the wild type is significant at <i>p</i><0.05.</p

Western blot analysis of CLU isoform.

<p>(A) Electrophoresis of stromal proteins from spleen and mesenteric lymph nodes (MLN) was performed in reducing and non-reducing conditions. Immunopositive bands mobility corresponds to secreted CLU isoform (sCLU). (B) Quantitative comparison of splenic sCLU expression in wild type (WT) and LTβR-deficient (LTβR-KO) mice. Data is normalized to the average WT expression and represented as mean±SD. * – Difference from the wild type is significant at <i>p</i><0.05.</p

Interfollicular channel region of mouse mesenteric lymph node.

<p>MLN was stained with anti-clusterin and ER-TR7 antibodies after immunization with SRBC. Note clear immunopositivity of conduits (arrows) for clusterin. Lower row represents the close up of the indicated square region. Data is representative of at least 2 experiments. Scale bar: 100 µm.</p

Cellular and tissue distribution of clusterin.

<p>Immunohistochemical staining of wild type spleen (A) and MLN (B) after immunization with SRBC. (C) Shows an image of clusterin-positive spleen stromal cell under high power (original magnification 630×), showing that clusterin is located near cell membrane. (D) Double staining for MRC marker ER-TR7 (green) and clusterin (red). Ubiquitous presence of clusterin-positive cells can be seen in all stromal compartments except for splenic marginal zone (D), with the brightest staining seen in germinal centers. Scale bar: 100 µm. GC – germinal center, CA – central arteriole, RP – red pulp, F – follicle, arrows – high endothelial venules, MZ – marginal zone. Data is representative of at least 2 experiments.</p

Changes in sCLU tissue distribution in spleen and MLN of WT mice at day 8 after immunization with SRBC.

<p>(A) Immunohistochemical staining of WT spleen cryosections showing accumulation of clusterin in the light zones of germinal centers (GC) upon immunization. (B) No changes in clusterin tissue distribution and quantity can be seen in MLN of WT mice after SRBC immunization. Data is representative of at least 2 independent experiments. Scale bar: 100 µm.</p