Figure 3

Reduction in disialyl-T structures on podoplanin in the lymph nodes of DKO mice. (A) Relative transcript levels of St6galnac1, St6galnac2, St6galnac3, and St6galnac4 in WT lymph nodes (n = 4). Hprt transcript levels were used for normalization. (B) Western blotting of podoplanin expression using the lysates of mesenteric lymph nodes in WT, 3KO, 4KO, and DKO mice. (C) Immunofluorescence staining of podoplanin (PDPN, green) and MALII lectin (red) in 3KO and DKO mesenteric lymph nodes. (D) Western blotting of podoplanin and MALII blotting using immunoprecipitated podoplanin from the lysates of mesenteric lymph nodes in WT, 3KO, 4KO, and DKO mice. Control rat IgG was used for detecting the unspecific bands. The immunoprecipitated samples were detected using an anti-podoplanin antibody and MALII lectin. (E) Western blots indicate Clec-2 levels in the lysates of platelets isolated from WT, 3KO, 4KO, and DKO peripheral blood. (F) Representative immunofluorescence staining images of VE-cadherin (red) and peripheral node addressin (PNAd, green) in DKO versus littermate control (3KO). (G,H) HEV area (G) and the area ratio of VE-cadherin/HEV (H) quantified using the results of immunofluorescence staining shown in Fig. 3F. HEV area was quantified by measuring the PNAd signal-positive area. 3KO: n = 3 (HEV: n = 271), DKO: n = 3 (HEV: n = 153), two-tailed Mann–Whitney’s rank test, n.s.; not significant, ***p < 0.001. LN Lymph node, IP immunoprecipitation, PDPN podoplanin.