Figure 5.
ENG protein expression in normal and HHT endothelial cells. (A) Schematic representations of ENG protein. (i) Domain organization of the ENG protein ectodomain in its BMP-binding homodimeric conformation, in relation to its genomic DNA origin, with OR domains highlighted in purple and ZP domains in blue. Q436 is distal to OR domains OR1 and OR2, but proximal to intermolecular dimerization cysteines C516 and C582 located within and after the ZP-C domain, respectively.72 For simplicity, the single-spanning C-terminal transmembrane domain of ENG (residues 587-611) is not shown. (ii) “Predicted” PTC-truncated ENG Q436X and R93X proteins in relation to the domains and SN6h epitope of wild-type ENG.72,83 (iii) ENG Q436X open reading frames from methionine 1 (M1) to PTC, and M445 to natural stop codon. (B) Pulse-chase experiments in which BOEC lysates were size fractionated on a reducing gel, and overexposed to optimize detection of minor, smaller bands. The approximate position of the Q436X protein that should be detectable by the 35S-labeled SN6h antibody is indicated by a gray arrow in BOECs heterozygous for (i) ENG Q436X, (ii) ENG R93X, (iii) SMAD4 Q368X, and (iv) ACVRL1 E391X. Note, no difference in patterns of sub–full length proteins in HHT BOECs, providing no evidence for a Q436X-specific truncated protein product at expected size based on truncation at the PTC (for full gel data see supplemental Figure 10). (C) Comparison of maturation patterns of full-length ENG protein after pulse-chase in BOECs heterozygous for (i-iv) ENG+/+ (wild-type) controls, and (v-vi) ENG+/PTC BOECs: (i) control A; (ii) control B; (iii) SMAD4 Q368X; (iv) ACVRL1 E391X, (v) exon 10 ENG Q436X, and (vi) exon 3 ENG R93X. (D) Densitometry analyses of the immunoblots in panel C. (i) Lower band (immature ENG) densitometry values across the 4 chase time points. By 2-way ANOVA, time accounted for 72.5% of variation (P < .0001), ENG vs non-ENG genotypes for 1.8% (P = .049), and the interaction term for 0.98% (P = .092). (ii) Upper band (mature ENG) densitometry values across the 4 chase time points. By 2-way ANOVA, time accounted for 64.3% of variation (P < .0001), ENG vs non-ENG genotypes for 1.4% (P = .54), and the interaction term for 3.5% (P = .26).

ENG protein expression in normal and HHT endothelial cells. (A) Schematic representations of ENG protein. (i) Domain organization of the ENG protein ectodomain in its BMP-binding homodimeric conformation, in relation to its genomic DNA origin, with OR domains highlighted in purple and ZP domains in blue. Q436 is distal to OR domains OR1 and OR2, but proximal to intermolecular dimerization cysteines C516 and C582 located within and after the ZP-C domain, respectively.72 For simplicity, the single-spanning C-terminal transmembrane domain of ENG (residues 587-611) is not shown. (ii) “Predicted” PTC-truncated ENG Q436X and R93X proteins in relation to the domains and SN6h epitope of wild-type ENG.72,83 (iii) ENG Q436X open reading frames from methionine 1 (M1) to PTC, and M445 to natural stop codon. (B) Pulse-chase experiments in which BOEC lysates were size fractionated on a reducing gel, and overexposed to optimize detection of minor, smaller bands. The approximate position of the Q436X protein that should be detectable by the 35S-labeled SN6h antibody is indicated by a gray arrow in BOECs heterozygous for (i) ENG Q436X, (ii) ENG R93X, (iii) SMAD4 Q368X, and (iv) ACVRL1 E391X. Note, no difference in patterns of sub–full length proteins in HHT BOECs, providing no evidence for a Q436X-specific truncated protein product at expected size based on truncation at the PTC (for full gel data see supplemental Figure 10). (C) Comparison of maturation patterns of full-length ENG protein after pulse-chase in BOECs heterozygous for (i-iv) ENG+/+ (wild-type) controls, and (v-vi) ENG+/PTC BOECs: (i) control A; (ii) control B; (iii) SMAD4 Q368X; (iv) ACVRL1 E391X, (v) exon 10 ENG Q436X, and (vi) exon 3 ENG R93X. (D) Densitometry analyses of the immunoblots in panel C. (i) Lower band (immature ENG) densitometry values across the 4 chase time points. By 2-way ANOVA, time accounted for 72.5% of variation (P < .0001), ENG vs non-ENG genotypes for 1.8% (P = .049), and the interaction term for 0.98% (P = .092). (ii) Upper band (mature ENG) densitometry values across the 4 chase time points. By 2-way ANOVA, time accounted for 64.3% of variation (P < .0001), ENG vs non-ENG genotypes for 1.4% (P = .54), and the interaction term for 3.5% (P = .26).

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