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. 2021 Jan 28;31(7):773–790. doi: 10.1038/s41422-020-00464-8

Fig. 7. The COMP EGF2 domain ameliorates AAA formation.

Fig. 7

a Upper panel, schematics illustrating the COMP constructs used to map the corresponding domains (N-terminus, EGF, type III, and C-terminus) that bind to the AT1a N-terminus. Presence or absence of binding between COMP domains and the AT1a N-terminus is indicated by + or –, respectively. Lower panel, mammalian two-hybrid analysis of the interaction between COMP and the AT1a N-terminus. COS-7 cells were transiently transfected with various domains of COMP cloned into pACT, together with the AT1a N-terminus cloned into pBIND. Cells were lysed after 48 h, and the luciferase activity was determined. The data are presented as the means ± SEM of 3 independent experiments performed in triplicate. *P < 0.05 by Two-way ANOVA followed by the Bonferroni test. b Upper panel, purified MBP-fusion proteins of the COMP N-terminus (MBP-COMP-N), EGF domain (MBP-COMP-EGF), Type III repeat (MBP-COMP-Type III) and C-terminus (MBP-COMP-C) were immobilized on amylose resin and incubated with the purified GST-fused N-terminus of AT1a. Trapped proteins were examined by immunoblotting with an anti-GST antibody. Purified MBP (first lane) was used as a control. Lower panel, western blot analysis of MBP-COMP-N, MBP-COMP-EGF, MBP-COMP-Type III and MBP-COMP-C. c Mammalian two-hybrid assay of the AT1a N-terminus with various EGF domains of COMP. The data are presented as the means ± SEM of 3 independent experiments performed in triplicate. One-way ANOVA followed by the Bonferroni test, *P < 0.05 vs pACT. d Representative images of the aortic morphology in 5-month-old male ApoE–/– mice periadventitially infected with Ad-GFP (Control) or Ad-EGF2 (COMP-EGF2) followed by 28 days of AngII infusion. e Incidence of AngII-induced AAA in ApoE–/– mice periadventitially infected with Ad-GFP (8/11) or Ad-EGF2 (4/15). *P < 0.05 by χ2 test. f The maximal abdominal aortic diameters of ApoE–/– mice periadventitially infected with Ad-GFP or Ad-EGF2. *P < 0.05 by Mann-Whitney test. g Representative images of VVG staining and quantification of elastin degradation. n = 9; *P < 0.05 by Mann-Whitney test. Scale bar, 50 μm.