Figure 1. Murine Spns2 is expressed by endothelial cells but not RBC.

(A) Expression of Spns2 mRNA by the indicated cell populations, expressed relative to hypoxanthine-guanine phosphoribosyltransferase (Hprt) transcript, assessed by RT-qPCR. Ter119⁺CD71⁺ immature RBC (iRBC) were sorted from bone marrow; CD31⁺gp38⁺ lymphatic endothelial cells (LEC), CD4⁺CD62L^hi T cells (CD4), CD8⁺CD62L^hi T cells (CD8), and CD19⁺CD62L^hi B cells (CD19) were sorted from lymph nodes. Data compile 3 experiments with mice on a B6 background.

(B) Expression of Spns2 protein by the indicated cell populations, assessed by Western blot. RBC were isolated from blood by differential centrifugation; CD31⁺ endothelial cells (EC) were isolated from heart and lung by magnetic bead enrichment; and CD4⁺ T cells, CD8⁺ T cells, and CD19⁺ B cells were isolated from lymph nodes by magnetic bead enrichment. Data are representative of 3 experiments with mice on a B6 background.

(C) Spns2-targeted allele. SA: splice acceptor; pA: polyadenylation signal.

(D-F) Efficiency of Spns2 deletion in Spns2^f/fTie2-Cre⁺ mice.

(D) PCR for Spns2 exon 3 in genomic DNA from sorted Spns2^f/fTie2Cre⁺(Δ) or littermate control (Ctrl) cells. Littermate controls maintained one or two intact alleles of Spns2. RBC progenitors were isolated from bone marrow. Hematopoietic stem and progenitor cells (HSPC) were defined as Lin⁻IL7Rα⁻c-Kit⁺Sca1⁺; common myeloid progenitors (CMP) as Lin⁻IL7Rα⁻c Kit⁺Sca1⁻FcγR^loCD34⁺; and megakaryocyte erythroid progenitors (MEP) as Lin⁻IL7Rα⁻c-Kit⁺Sca1⁻FcγR^loCD34⁻. Lymphatic endothelial cells (LEC), defined as CD45⁻CD31⁺gp38⁺, and fibroblastic reticular cells (FRC), defined as CD45⁻CD31⁻gp38⁺ were isolated from lymph nodes. Data are representative of at least 2 experiments. (E) Spns2 mRNA assessed by RT-qPCR of transcripts from sorted LEC and FRC. Data compile 7 pairs of mice analyzed in 7 experiments for LEC and 3 pairs of mice analyzed in 3 experiments for FRC. NS, no signal. (F) Spns2 protein assessed by Western blot of mixed heart and lung endothelial cells (EC). Data are representative of 2 experiments. See also Fig. S1.