Figure 8.
Blockade of uPA results in reduced CCL21-ΔC levels in skin-dLNs. (A and B) Western blot–based comparison of CCL21 proteoforms in steady-state LNs and ear skin. (A) Representative western blots performed on tissue protein extracts and (B) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 western blots from independent experiments. (C) Representative western blot of CCL21 performed on LN extracts, LN eluates, and serum. (D) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN extracts, LN eluates, or serum. Antibody clone MAB457 detects full-length CCL21 only, whereas clone AF457 detects both full-length CCL21 and CCL21-ΔC. Data from n = 3 mice are shown. n.d., not detected. (E) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN eluates from WT and uPAmut mice. Data from n = 4–5 mice are shown, Student’s t test. n.d., not detected. (F and G) Protein extracts were prepared from LNs of PBS-perfused WT and uPAmut mice and used for ELISA-based quantification of (F) plasminogen and (G) assessment of plasmin activity (colorimetric assay). Pooled data from n = 6 mice per group are shown as mean ± SEM. Student’s t test. (H and I) Analysis of CCL21 levels in LNs of WT and uPAmut mice. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. Images were subjected to AI-based tissue segmentation for differentiating between the T cell zone and B cell follicles/SCS. (H) Representative images from the immunofluorescent staining performed on LNs of WT and uPAmut mice. Scale bar: 100 μm. (I) Quantification of CCL21 staining intensity observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. Source data are available for this figure: SourceData F8.

Blockade of uPA results in reduced CCL21-ΔC levels in skin-dLNs. (A and B) Western blot–based comparison of CCL21 proteoforms in steady-state LNs and ear skin. (A) Representative western blots performed on tissue protein extracts and (B) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 western blots from independent experiments. (C) Representative western blot of CCL21 performed on LN extracts, LN eluates, and serum. (D) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN extracts, LN eluates, or serum. Antibody clone MAB457 detects full-length CCL21 only, whereas clone AF457 detects both full-length CCL21 and CCL21-ΔC. Data from n = 3 mice are shown. n.d., not detected. (E) ELISA-based quantification of full-length CCL21 and CCL21-ΔC present in LN eluates from WT and uPAmut mice. Data from n = 4–5 mice are shown, Student’s t test. n.d., not detected. (F and G) Protein extracts were prepared from LNs of PBS-perfused WT and uPAmut mice and used for ELISA-based quantification of (F) plasminogen and (G) assessment of plasmin activity (colorimetric assay). Pooled data from n = 6 mice per group are shown as mean ± SEM. Student’s t test. (H and I) Analysis of CCL21 levels in LNs of WT and uPAmut mice. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. Images were subjected to AI-based tissue segmentation for differentiating between the T cell zone and B cell follicles/SCS. (H) Representative images from the immunofluorescent staining performed on LNs of WT and uPAmut mice. Scale bar: 100 μm. (I) Quantification of CCL21 staining intensity observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. Source data are available for this figure: SourceData F8.

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