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. 2016 Nov 14;213(12):2773–2791. doi: 10.1084/jem.20160287

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© 2016 Zamora-Pineda et al.

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Figure 5. — SPL deficiency in endothelial cells does not block thymic egress. (A) Immunofluorescence was performed on frozen thymic sections from SPL^F and SPL^VEKO mice using CD31, SPL antibody, and DAPI (blue) followed by staining with fluorophore-conjugated secondary antibodies. Dotted lines delineate a blood vessel. Bar, 100 µm. Images shown are representative of five thymuses analyzed. (B) Western blot of whole-cell extracts of thymic endothelial cells (CD31⁺ CD45⁻ CK8⁻) isolated from SPL^F and SPL^VEKO mice. GAPDH was used as a loading control. (C) Relative intensity of Western blot bands of SPL/GAPDH. The results shown are representative of two independent experiments. (D) Absolute number of CD4SP and mature CD4SP T cells from SPL^F and SPL^VEKO mice. (E) Absolute numbers of CD4⁺ and CD8⁺ T cells in the blood, mesenteric LN, and spleen. (D and E) Graphs represent a compilation of four independent experiments. SPL^F, n = 7; SPL^VEKO, n = 12. (F) S1P₁ surface abundance on mature CD4SP T cells in representative SPL^F and SPL^VEKO mice. The gray histogram shows a negative isotype control. (G) Quantification of mean fluorescence intensity (MFI) of S1P₁ (SPL^F, n = 3; SPL^VEKO, n = 3). The results shown are representative of two independent experiments. (D, E, and G) Data are shown as mean ± SD for two-tailed unpaired Student’s t tests.