Figure 1
Figure 1. Interaction between Prox1 and COUP-TFII proteins and their colocalization in LECs. (A) Sequence alignments of human Lrh1, SF-1, zebrafish ff1b, and COUP-TFII show that COUP-TFII harbors the LLLRLP sequence motif for interaction with Prox1.15–17 DBD indicates DNA-binding domain; and LBD, ligand-binding domain. (B) Mammalian 2-hybrid assay demonstrating a physical interaction of Prox1 and COUP-TFII proteins. Expression constructs for human full-length Prox1 and mouse COUP-TFII (67∼414 aa) proteins fused with either BD (GAL4-DNA–binding domain) or AD (VP16-activation domain) were transfected with a reporter vector into HEK293 cells and their activities on the expression of the luciferase reporter were measured after 48 hours. (C,D) Coimmunoprecipitation of Prox1 and COUP-TFII proteins; whole-cell lysates (WCL) from HEK293 cells that were transfected with the control or expression vector of Flag-Prox1 and/or HA-COUP-TFII were immunoprecipitated (IP) with anti-HA (C) or anti-Flag (D) antibodies and immunoblotted (IB) with anti-Flag and anti-HA antibodies, respectively. Endogenous Prox1 from HepG2 (E) or primary human LEC cells (F) were immunoprecipitated using an anti–COUP-TFII antibody in the absence or presence of ethidium bromide (EtBr, 100 μg/mL). IgG, a control isotype antibody. (G) Coexpression of Prox1 and COUP-TFII in developing and postdevelopmental lymphatic vessels. i-iv: COUP-TFII is expressed in Prox1-positive ECs of the cardinal vein (arrowhead) and in sprouting LECs of mouse embryo (E11.5). v-viii: Enlarged images of 2 newly formed lymphatic vessels and budding LECs (shown in boxes in ii and iii). Neither Prox1 nor COUP-TFII is expressed in the dorsal aorta (arrow). Bar, 100 μm. Human foreskin sections were stained for LYVE-1/COUP-TFII (ix-xii) and Prox1/COUP-TFII (xiii-xvi) and Prox1- or LYVE-positive LECs (arrowheads) clearly express COUP-TFII, whereas venous ECs (arrows) express only COUP-TFII. Expression of COUP-TFII and Prox1 in cultured LECs and BECs isolated from human foreskins: LECs express both Prox1 and COUP-TFII (xvii-xx) and the COUP-TFII expression levels are comparable in Prox1-positive LECs (arrowheads) versus Prox1-negative BECs (arrows) in a mixed culture of both cell types (xxi-xxiv). Bars, 100 μm.

Interaction between Prox1 and COUP-TFII proteins and their colocalization in LECs. (A) Sequence alignments of human Lrh1, SF-1, zebrafish ff1b, and COUP-TFII show that COUP-TFII harbors the LLLRLP sequence motif for interaction with Prox1.15-17  DBD indicates DNA-binding domain; and LBD, ligand-binding domain. (B) Mammalian 2-hybrid assay demonstrating a physical interaction of Prox1 and COUP-TFII proteins. Expression constructs for human full-length Prox1 and mouse COUP-TFII (67∼414 aa) proteins fused with either BD (GAL4-DNA–binding domain) or AD (VP16-activation domain) were transfected with a reporter vector into HEK293 cells and their activities on the expression of the luciferase reporter were measured after 48 hours. (C,D) Coimmunoprecipitation of Prox1 and COUP-TFII proteins; whole-cell lysates (WCL) from HEK293 cells that were transfected with the control or expression vector of Flag-Prox1 and/or HA-COUP-TFII were immunoprecipitated (IP) with anti-HA (C) or anti-Flag (D) antibodies and immunoblotted (IB) with anti-Flag and anti-HA antibodies, respectively. Endogenous Prox1 from HepG2 (E) or primary human LEC cells (F) were immunoprecipitated using an anti–COUP-TFII antibody in the absence or presence of ethidium bromide (EtBr, 100 μg/mL). IgG, a control isotype antibody. (G) Coexpression of Prox1 and COUP-TFII in developing and postdevelopmental lymphatic vessels. i-iv: COUP-TFII is expressed in Prox1-positive ECs of the cardinal vein (arrowhead) and in sprouting LECs of mouse embryo (E11.5). v-viii: Enlarged images of 2 newly formed lymphatic vessels and budding LECs (shown in boxes in ii and iii). Neither Prox1 nor COUP-TFII is expressed in the dorsal aorta (arrow). Bar, 100 μm. Human foreskin sections were stained for LYVE-1/COUP-TFII (ix-xii) and Prox1/COUP-TFII (xiii-xvi) and Prox1- or LYVE-positive LECs (arrowheads) clearly express COUP-TFII, whereas venous ECs (arrows) express only COUP-TFII. Expression of COUP-TFII and Prox1 in cultured LECs and BECs isolated from human foreskins: LECs express both Prox1 and COUP-TFII (xvii-xx) and the COUP-TFII expression levels are comparable in Prox1-positive LECs (arrowheads) versus Prox1-negative BECs (arrows) in a mixed culture of both cell types (xxi-xxiv). Bars, 100 μm.

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