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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: J Immunol. 2014 Feb 3;192(5):2133–2142. doi: 10.4049/jimmunol.1301286

Figure 5. Presence of appropriately sulfated heparan sulfate in lymphatic endothelial conditioned medium is required for binding of CCL21 to bone marrow-derived DC.

Figure 5

Conditioned medium (CM) was collected from hLEC transfected with either control siRNA (siDS) or siRNA targeting the HS biosynthetic enzymes Ndst1 or XylT2. Cytospin samples of day 9 BMDC were incubated with the different CM. Binding of CCL21 in the CM to CCR7 on BMDC was detected by proximity ligation assay (PLA). Representative merged images showing PLA signal (red) and nuclear DAPI stain (blue) were taken by fluorescence microscopy (400 ×). Signal for BMDC incubated in the vehicle control medium alone (EBM2 containing 5% horse serum; which served as the basal medium for all hLEC CM) is shown on the upper left panel. PLA signal from each field was quantified and indexed to total nuclear area within the same field. The average of at least 5 random fields from each group was included for analysis, with mean data normalized to control signal (CM-siDS) (see graph below). *P < 0.05, #P < 0.01, as compared to CM-siDS group.