(A) ProS structure: gamma carboxyglutamic acid (Gla) domain, thrombin-sensitive region (TSR), EGF-like repeats, and SHBG domain containing 2 laminin G repeats. (B) Mouse Pros1 locus with exon 1 encoding the ProS signal peptide (sig pep). (C) Targeting vector: Exons 11–15 were flanked by loxP sites. PstI (blue), BglI (black), and HpaI (green) sites used to characterize targeting. (D) Southern blot of DNA from an ES cell clone, using Pstl and BglI digests and the 5′ and 3′ external probes indicated in C (left 2 lanes). PstI diagnostic digest of genomic DNA from WT (+/+), heterozygous (fl/+), and homozygous (fl/fl) floxed mice (middle 3 lanes). Crossing floxed mice to the EIIA-Cre general deleter generated a KO Pros1 allele (far right lane). HpaI-digested genomic DNA from WT and KO/+ mice blotted and hybridized with the 5′ probe (right 2 lanes). (E) qPCR of Pros mRNA from E17.5 WT (n = 8) and KO (n = 6) embryos; mean ± 1 SD. (F) Western blot of protein from WT (+/+) and heterozygous Pros1^+/– (KO/+) mice, with an anti-ProS antibody generated against the amino terminus of the protein (upper blot) and anti–β-actin (lower blot). ProS appears as a full-length protein (upper band) and as a thrombin-cleaved form (lower band). Both lanes were run on the same gel but were noncontiguous. Transcripts from nontargeted aminoterminal exons 4–5 are present in Pros1^+/– heterozygotes (E) but apparently do not code for a stable protein. (See also Figure 9A for a related immunoblot.)