FIGURE 2.

Expression and secretion of GT-8 fibrillin-1. A, left panel: using 3′ Fbn1 primers, quantitative PCR analyses were used to quantitate expression of Fbn1. The -fold differences were calculated compared with average levels in wild-type mice. mRNA from skin of Fbn1^−/− mice was used as a control. Levels of 3′ Fbn1 in homozygous GT-8 mice were not significantly different from −/− mice, indicating that recombination of mutant alleles occurred in most, if not all, cells. Right panel: quantitative PCR of Fbn2 mRNA levels showed no significant differences between wild-type, heterozygous, and homozygous GT-8 mice. mRNA from Fbn2^−/− mice was included as a control. B, Western blotting of proteins secreted into the medium by skin fibroblast cultures was performed using pAb 9543 (anti-fibrillin-1) (left panel) and anti-GFP (right panel). Blotting with anti-GFP established the identity of the truncated fibrillin-1. These fibroblast cultures were established from a single litter. Fibroblasts from pups 17, 18, 20, and 22 secreted only wild-type fibrillin-1, whereas fibroblasts from pups 16, 19, and 21 secreted both wild-type and eGFP-tagged and -truncated fibrillin-1. Blotting with pAb 9543 demonstrated that the wild-type and truncated fibrillin-1 were present in approximately equal amounts in the medium of heterozygous cultures. C, quantitation of fibrillin-1 was determined by sandwich ELISA. Antibody pairs were directed toward the N-terminal half of fibrillin-1, so that both wild-type and truncated fibrillin-1 would be captured. Skin fibroblast cultures from a litter of GT8 mice were plated (100,000 cells/well) and incubated for 72 h. The mean concentration (and standard deviation) of all cell lines for each genotype is shown (n = 1 for +/+; n = 4 for GT-8/+; n = 5 for GT-8/GT-8). Fibrillin-1 was detected in all cell lines and did not vary significantly by genotype (p = 0.56). Fibrillin-2 was not detectable in the cell culture media (data not shown).