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. 2004 Mar 1;164(5):769–779. doi: 10.1083/jcb.200307137

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Copyright © 2004, The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 3. — PMA-stimulated shedding of EGFR ligands in adam ^−/− cells. (A) The first panel shows an explanatory diagram depicting how increases in ectodomain shedding of different EGFR ligands from a given well are presented in the remaining panels of this figure. Shedding stimulated by 20 ng/ml PMA for 1 h is calculated as the percent increase in AP activity over constitutive shedding in the same well for 1 h. The next panels only show the PMA-dependent increase in shedding over constitutive levels for each EGFR ligand and each adam ^−/− cell type. Data from primary mEFs (yellow bars) are compiled from separate experiments using cells from three or more litters for each adam ^−/− mouse line. Only adam10 ^−/− and control adam10 ^+/− cells were immortalized (blue bars). Overall, at least four separate wells were evaluated per EGFR ligand. The results indicate that ADAM17 is the major stimulated sheddase for TGFα, amphiregulin, epiregulin, and HB-EGF. ADAMs 9, 12, or 15 (or a combination of two or more of these ADAMs) also contribute to stimulated epiregulin shedding. On the other hand, the shedding of betacellulin and EGF is only weakly stimulated by PMA. Because the increase in stimulated shedding is small, no statistically significant differences in stimulated shedding of betacellulin or EGF was seen in adam ^−/− cells compared with wild-type controls. (B) Histological analysis of sectioned hematoxylin and eosin–stained eyes and eyelids (A–D), aortic valves (E–H), pulmonic valves (I–L), and tricuspid valves (M–P) of newborn wild-type (A, E, I, and M), adam9/12/15 ^−/− (B, F, J, and N), adam17 ^−/− (C, G, K, and O), and adam9/12/15/17 ^−/− (D, H, L, and P) mice. Eyelids of wild-type and adam9/12/15 ^−/− mice are closed at birth (A and B), whereas those of adam17 ^−/− and adam9/12/15/17 ^−/− mice are open (C and D). The aortic, pulmonic, and tricuspid valves of adam9/12/15 ^−/− mice (F, J, and N) resemble those of wild-type mice (E, I, and M, respectively), whereas these valves are thickened and misshapen in adam17 ^−/− (G, K, and C) and adam9/12/15/17 ^−/− quadruple knockout mice (H, L, and P). The valve defects in adam9/12/15/17 ^−/− quadruple knockout mice, which also include thickened and misshapen mitral valves (not depicted), are comparable to those in adam17 ^−/− mice. Eyelids in A–D marked by red arrows, heart valves in E–P marked by yellow arrows. Bar (E–P), 100 μm.

Figure 3. — PMA-stimulated shedding of EGFR ligands in adam ^−/− cells. (A) The first panel shows an explanatory diagram depicting how increases in ectodomain shedding of different EGFR ligands from a given well are presented in the remaining panels of this figure. Shedding stimulated by 20 ng/ml PMA for 1 h is calculated as the percent increase in AP activity over constitutive shedding in the same well for 1 h. The next panels only show the PMA-dependent increase in shedding over constitutive levels for each EGFR ligand and each adam ^−/− cell type. Data from primary mEFs (yellow bars) are compiled from separate experiments using cells from three or more litters for each adam ^−/− mouse line. Only adam10 ^−/− and control adam10 ^+/− cells were immortalized (blue bars). Overall, at least four separate wells were evaluated per EGFR ligand. The results indicate that ADAM17 is the major stimulated sheddase for TGFα, amphiregulin, epiregulin, and HB-EGF. ADAMs 9, 12, or 15 (or a combination of two or more of these ADAMs) also contribute to stimulated epiregulin shedding. On the other hand, the shedding of betacellulin and EGF is only weakly stimulated by PMA. Because the increase in stimulated shedding is small, no statistically significant differences in stimulated shedding of betacellulin or EGF was seen in adam ^−/− cells compared with wild-type controls. (B) Histological analysis of sectioned hematoxylin and eosin–stained eyes and eyelids (A–D), aortic valves (E–H), pulmonic valves (I–L), and tricuspid valves (M–P) of newborn wild-type (A, E, I, and M), adam9/12/15 ^−/− (B, F, J, and N), adam17 ^−/− (C, G, K, and O), and adam9/12/15/17 ^−/− (D, H, L, and P) mice. Eyelids of wild-type and adam9/12/15 ^−/− mice are closed at birth (A and B), whereas those of adam17 ^−/− and adam9/12/15/17 ^−/− mice are open (C and D). The aortic, pulmonic, and tricuspid valves of adam9/12/15 ^−/− mice (F, J, and N) resemble those of wild-type mice (E, I, and M, respectively), whereas these valves are thickened and misshapen in adam17 ^−/− (G, K, and C) and adam9/12/15/17 ^−/− quadruple knockout mice (H, L, and P). The valve defects in adam9/12/15/17 ^−/− quadruple knockout mice, which also include thickened and misshapen mitral valves (not depicted), are comparable to those in adam17 ^−/− mice. Eyelids in A–D marked by red arrows, heart valves in E–P marked by yellow arrows. Bar (E–P), 100 μm.